Muteins of a1m lipocalin and method of production therefor

ABSTRACT

The present disclosure relates to a collection of novel muteins derived from human α1m (or a1m) polypeptide or a functional homologue thereof. The disclosure further refers to a α1m mutein capable of specifically binding to one or more targets other than a target to which wild-type α1m binds. The disclosure also relates to a method for producing such collection of muteins and a method for isolating a mutein capable of binding one or more such non-natural targets of wild-type α1m polypeptide. These aspects are made possible due to, e.g, the structural elucidation of α1m disclosed herein by the present inventors, an appreciation of ligand-binding sights thereof and, hence, an understanding of which amino acid positions are most suitable for mutagenesis for re-engineering specificity and affinity for any given target while maintaining the secondary and/or tertiary structure of a1m.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/375,188, filed Jul. 29, 2014, which is a National Stage applicationof PCT/EP2013/051962, filed Jan. 31, 2013, which claims priority fromU.S. Provisional Application No. 61/592,843, filed Jan. 31, 2012.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 11, 2016, isnamed 029029-0154_SL.txt and is 97,042 bytes in size.

BACKGROUND

Proteins that selectively bind to selected targets by way ofnon-covalent interaction play a crucial role as reagents inbiotechnology, medicine, bioanalytics as well as in the biological andlife sciences in general. Antibodies, i.e. immunoglobulins, are aprominent example of this class of proteins. Despite the manifold needsfor such proteins in conjunction with recognition, binding and/orseparation of ligands/targets, almost exclusively immunoglobulins arecurrently used.

Additional proteinaceous binding molecules that have antibody-likefunctions are certain members of the lipocalin family, which havenaturally evolved to endogenously bind ligands. Lipocalins occur in manyorganisms, including vertebrates, insects, plants and bacteria. Membersof the lipocalin protein family (Pervaiz, S., & Brew, K. (1987) FASEB J.1, 209-214) are typically small, secreted proteins and have a singlepolypeptide chain. They are characterized by a range of differentmolecular-recognition properties: their ability to bind various,principally hydrophobic molecules (such as retinoids, fatty acids,cholesterols, prostaglandins, biliverdins, pheromones, tastants, andodorants), their binding to specific cell-surface receptors and theirformation of macromolecular complexes. Although they have, in the past,been classified primarily as transport proteins, it is now clear thatthe lipocalins fulfill a variety of physiological functions. Theseinclude roles in retinol transport, olfaction, pheromone signaling, andthe synthesis of prostaglandins. The lipocalins have also beenimplicated in the regulation of the immune response and the mediation ofcell homeostasis (reviewed, for example, in Flower, D. R. (1996)Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys.Acta 1482, 9-24).

α₁-Microglobulin (α₁m)—also known as protein HC, α₁-glycoprotein orα₁-microglyco-protein—is a 26 kDa glycoprotein with 184 amino acidresidues which is abundant in blood plasma, urine, and connective tissueof humans as well as vertebrate animals (Åkerström, B., Lögdberg, L.,Berggärd, T., Osmark, P., and Lindqvist, A. (2000) Biochim Biophys Acta1482, 172-184). Based on characteristic amino acid sequence motifs, α₁mhas been assigned a member of the lipocalin family (Pervaiz, S., andBrew, K. (1987) FASEB J 1, 209-214; Pervaiz, S., and Brew, K. (1985)Science 228, 335-337), although this has been done without knowing thethree-dimensional structure of this biomolecule. Natural a1m from urineand plasma is heterogeneous in size and charge and has a characteristicyellow-brown color Åkerström, B., and Berggärd, I. (1979) Eur J Biochem101, 215-223; Berggärd, T., Cohen, A., Persson, P., Lindqvist, A.,Cedervall, T., Silow, M., Thogersen, I. B., Jonsson, J. A., Enghild, J.J., and Åkerström, B. (1999) Protein Sci 8, 2611-2620), an attributethat also served to designate this lipocalin (kerström, B., Lögdberg,L., Berggärd, T., Osmark, P., and Lindqvist, A. (2000) Biochim BiophysActa 1482, 172-184). α1m is glycosylated at three sites: two complexcarbohydrates are N-linked to residues Asn17 and Asn96 while Thr5 isO-glycosylated (Ekström, B., Lundblad, A., and Svensson, S. (1981) Eur JBiochem 114, 663-666; Escribano, J., Lopex-Otin, C., Hjerpe, A., Grubb,A., and Mendez, E. (1990) FEBS Lett 266, 167-170). Since its initialidentification in humans (Ekström, B., Peterson, P. A., and I., B.(1975) Biochem Biophys Res Commun. 65, 1427-1433), α1m has beenassociated with various physiological processes includingimmunosuppression (Åkerström, B., Lögdberg, L., Berggärd, T., Osmark,P., and Lindqvist, A. (2000) Biochim Biophys Acta 1482, 172-184;Lögdberg, L., and Åkerström, B. (1981) Scand J Immunol 13, 383-390),lymphocyte stimulation, and also inhibition of lymphocyte cellproliferation (Wester, L., Michaelsson, E., Holmdahl, R., Olofsson, T.,and Åkerström, B. (1998) Scand J Immunol 48, 1-7) as well as neutrophilchemotaxis (Mendez, E., Fernandez-Luna, J. L., Grubb, A., andLeyva-Cobian, F. (1986) Proc Natl Acad Sci USA 83, 1472-1475).Furthermore, several biochemical activities have been ascribed to α1m,in particular in the context of heme and tryptophan metabolism (Olsson,M. G., Olofsson, T., Tapper, H., and Åkerström, B. (2008) Free Radic Res42, 725-736; Allhorn, M., Berggärd, T., Nordberg, J., Olsson, M. L., andÅkerström, B. (2002) Blood 99, 1894-1901) and with regard to reductaseand radical scavenging functions (Allhorn, M., Klapyta, A., andÅkerström, B. (2005) Free Radic Biol Med 38, 557-567; Åkerström, B.,Maghzal, G. J., Winterbourn, C. C., and Kettle, A. J. (2007) J Biol Chem282, 31493-31503). In addition, α1m serves as an important biomarker inclinical diagnostics for the monitoring of renal tubular dysfunction,renal toxicity, preeclampsia, and hepatitis E (Bolt, H. M., Lammert, M.,Selinski, S., and Bruning, T. (2004) Int Arch Occup Environ Health 77,186-190; Taneja, S., Sen, S., Gupta, V. K., Aggarwal, R., and Jameel, S.(2009) Proteome Sci 7, 39; Yu, H., Yanagisawa, Y., Forbes, M. A.,Cooper, E. H., Crockson, R. A., and MacLennan, I. C. (1983) J ClinPathol 36, 253-259; Devarajan, P., Krawczeski, C. D., Nguyen, M. T.,Kathman, T., Wang, Z., and Parikh, C. R. (2010) Am J Kidney Dis 56,632-642; Anderson, U. D., Olsson, M. G., Rutardottir, S., Centlow, M.,Kristensen, K. H., Isberg, P. E., Thilaganathan, B., Åkerström, B., andHansson, S. R. (2011) Am J Obstet Gynecol 204, 520 e521-525). Apart fromthese observations, no dedicated physiological ligand for the centralhydrophobic pocket of α1m—a characteristic feature of all lipocalinproteins (Flower, D. R. (1996) Biochem J 318, 1-14; Skerra, A. (2000)Biochim Biophys Acta 1482, 337-350)—could be identified so far.

Various PCT publications (e.g., WO 99/16873, WO 00/75308, WO 03/029463,WO 03/029471 and WO 2005/19256) disclose how muteins of variouslipocalins (e.g. tear lipocalin and hNGAL lipocalin) can be constructedto exhibit a high affinity and specificity against a target that isdifferent than a natural ligand of a wild type lipocalin. This can bedone by mutating certain positions of the lipocalin in a rationalmanner.

Despite the advances made with certain lipocalins in terms ofre-engineering their specificity, there remains a need for thegeneration of other lipocalin muteins that contain different bindingsites and alternative lipocalin scaffolds that can be used for suchgeneration. In view of the various potential applications for ligand- ortarget-binding proteins in the field of life sciences and biotechnology,the generation of muteins of yet other lipocalins would be desirable to,e.g., widen the spectrum of clinical targets against which lipocalinmuteins may bind. Without knowing the secondary and tertiary structureof a lipocalin and hence its potential binding sites, any rationalattempt to generate one or more muteins of that lipocalin to bind atarget of interest would be futile.

Accordingly, to meet said need, the present disclosure provides, forexample, the structural elucidation of the lipocalin scaffold human α₁mto create, e.g., a collection (i.e. library) of lipocalin muteinsincluding members (i.e. muteins) that have binding affinity andspecificity to at least one target other than a target to whichwild-type a1m binds. The present disclosure also provides lipocalinmuteins whose pocket or loop region may contain more than one bindingsite as, e.g., the central pocket (i.e. cavity) of the a1m polypeptideis wider than that in other lipocalins.

SUMMARY

The present disclosure can be characterized by the followingEmbodiments:

-   Embodiment 1. A collection of at least 10{circumflex over ( )}2    amino acid sequence members comprised of muteins of human a1m    polypeptide or a functional homologue thereof, wherein the amino    acid sequence of said members differs from human a1m polypeptide at    one or more of the sequence positions which correspond to the    sequence positions in the four peptide loops #1, #2, #3 and #4 of    human a1m polypeptide, and wherein the members of said collection    have at least 60% sequence identity with human a1m polypeptide.-   Embodiment 2. The collection of Embodiment 1, wherein at least one    of said members can specifically bind to at least one target other    than a target to which wild-type a1m binds and has no or no    substantial binding affinity for endogenous a1m target(s).-   Embodiment 3. The collection of Embodiment 1, wherein at least one    of said members can specifically bind to at least one target other    than the endogenous target(s) to which wild-type a1m binds and    retains substantial binding affinity for one or more endogenous a1m    target(s).-   Embodiment 4. The collection of Embodiment 1, wherein at least one    of said members can specifically bind to two targets other than a    target to which wild-type a1m binds and has no or no substantial    binding affinity for said endogenous a1m target, wherein said at    least one of said members has two binding sites.-   Embodiment 5. The collection of Embodiment 1 or 2, wherein a    target-binding site of said is within any or all of the four peptide    loops #1, #2, #3 and #4 of human a1m polypeptide.-   Embodiment 6. The collection of Embodiment 3, wherein two    target-binding sites are within any or all of the four peptide loops    #1, #2, #3 and #4 of human a1m polypeptide.-   Embodiment 7. The collection according to any of Embodiments 1 to 6,    wherein the amino acid sequence of said members differs from human    a1m polypeptide at one or more of the sequence positions which    correspond to the linear polypeptide sequence positions 29-48,    63-80, 89-100, and 115-129 of human a1m polypeptide.-   Embodiment 8. The collection according to any of Embodiments 1 to 6,    wherein the amino acid sequence of said members differs from human    a1m polypeptide at one or more of the sequence positions which    correspond to the linear polypeptide sequence positions 32-46,    66-72, 91-98, and 118-126 of human a1m polypeptide.-   Embodiment 9. The collection according to any of Embodiments 1 to 6,    wherein the amino acid sequence of said members differs from human    a1m polypeptide at one or more of the sequence positions which    correspond to the linear polypeptide sequence positions 34-37,    62-64, 97-99, 116-118, and 126-130 of human a1m polypeptide.-   Embodiment 10. The collection according to any one of Embodiments    7-9, wherein the amino acid sequence of said members further differs    from human a1m polypeptide at one or more of the sequence positions    which correspond to the linear polypeptide sequence positions 30,    47, 64, 73, 75, 77, 79, 90, 99, 116, and 128 of human a1m    polypeptide.-   Embodiment 11. The collection according to any one of Embodiments 1    to 10, wherein a nucleic acid molecule coding for each member is    operably fused thereto with a gene coding for the coat protein pIII    of a filamentous bacteriophage of the M13-family or for a fragment    of this coat protein.-   Embodiment 12. A collection of nucleic acid molecules, each    comprising a nucleotide sequence encoding a member comprised in the    collection of amino acid sequence members of any of Embodiments 1 to    10, wherein the amino acid sequence of said members has at least 60%    sequence homology with human mature a1m sequence and wherein said    members comprise at least one mutated amino acid residues at any    sequence position in the four peptide loops #1, #2, #3 and #4.-   Embodiment 13. A vector comprising the nucleic acid molecule of    Embodiment 12.-   Embodiment 14. The vector of Embodiment 12, which is a phagemid    vector.-   Embodiment 15. A host cell containing a nucleic acid molecule of    Embodiment 12 or a vector of Embodiment 13 or 14.-   Embodiment 16. A method of producing a mutein of human a1m    polypeptide or a functional homologue thereof, which mutein    specifically binds a target other than a target to which wild-type    a1m binds and has no or no substantial binding affinity for an    endogenous a1m target, comprising the steps of: (i) screening the    collection of any of Embodiments 1 to 11 with a target other than a    target to which wild-type a1m binds under conditions that allow    formation of a complex between (a) said target desired to be    specifically bound and (b) said collection, (ii) removing muteins of    the collection having no or no substantial binding affinity to said    target; and (iii) isolating the mutein specifically binding to said    target.-   Embodiment 17. A mutein derived from human a1m polypeptide or a    functional homologue thereof, wherein the mutein comprises at least    one mutated amino acid residues at any sequence position in the four    peptide loops #1, #2, #3 and #4, wherein said a1m or functional    homologue thereof has at least 60% sequence homology with human    mature a1m sequence, and wherein the mutein can bind a target other    than a target to which wild-type a1m binds and wherein the mutein    has no or no substantial binding affinity for endogenous a1m target.-   Embodiment 18. A mutein according to Embodiment 17, wherein the    mutein comprises at least one mutated amino acid residues at any    sequence position corresponding to sequence positions 29-48, 63-80,    89-100, and 115-129 of human mature a1m sequence.-   Embodiment 19. A mutein according to Embodiment 17, wherein the    mutein comprises at least one mutated amino acid residues at any    sequence position corresponding to the linear polypeptide sequence    positions 32-46, 66-72, 91-98, and 118-126 of human mature a1m    sequence.-   Embodiment 20. A mutein according to Embodiment 17, wherein the    mutein comprises at least one mutated amino acid residues at any    sequence position corresponding to the linear polypeptide sequence    positions 34-37, 62-64, 97-99, 116-118 and 126-130 of human mature    a1m sequence.-   Embodiment 21. A mutein according to any one of Embodiments 18-20,    wherein the mutein further comprises at least one mutated amino acid    residues at any sequence position corresponding to the linear    polypeptide sequence positions 30, 47, 73, 75, 77, 79 and 90 of    human mature a1m sequence.-   Embodiment 22. The mutein according to any one of Embodiments 17 to    21, wherein said mutein can specifically bind to two targets other    than a target to which wild-type a1m binds.-   Embodiment 23. The mutein according to Embodiment 22, wherein said    mutein has two binding sites.-   Embodiment 24. The mutein according to any one of Embodiments 17 to    21, wherein said mutein can specifically bind to Colchicine.-   Embodiment 25. The mutein according to any one of Embodiments 17 to    21, wherein said mutein can specifically bind to Lutetium (177Lu)    DOTA-TATE.-   Embodiment 26. An a1m crystal having space group P3₂21 and unit-cell    parameters a=b=66.72, c=80.26, a=b=90.0, g=120.0.-   Embodiment 27. A pharmaceutical composition comprising a mutein of    any one of Embodiments 17 to 25 and a pharmaceutically acceptable    carrier or excipient.-   Embodiment 28. A diagnostic composition comprising a mutein of any    one of Embodiments 17 to 25 and optionally means for diagnostic such    as a label or marker that is to be coupled, bound or complexed    with/to said lipocalin.

DESCRIPTION OF FIGURES

FIG. 1. Crystal structure of human a1m.

In FIG. 1A, secondary structure elements are shown as a cartoonpresentation (yellow, β-strands; pink, α-helices). The following aminoacid side chains are highlighted as sticks: the disulfide bridge(Cys72-Cys169), the position of the unpaired Cys34 in the native proteinthat gives rise to covalent crosslinking to other plasma proteins suchas IgA (Ser in the recombinant protein), the four residues assumed to beinvolved in chromophore binding (Ser/Cys34, Lys92, Lys118, Lys130), twoN-glycosylation sites (Asn17 and Asn96), and the pair of His122 andHis123 that participate in Ni²⁺ complexation (see below). The four loopswhich connect neighboring β-strands at the open end of theeight-stranded β-barrel and form the entry to the characteristic ligandpocket are labeled #1 to #4. Strand I is not part of the β-barrel butconstitutes an extended segment spanning the distance between the longα-helix that is attached to the side of the β-barrel and the C-terminaldisulfide bond.

The FIG. 1B depicts the stereo view of a section through theelectrostatic potential surface of a1m (from −10 kBT/e, red, to +10kBT/e, blue), illustrating the deep and positively charged ligandpocket.

In FIG. 1C, the Ni²⁺ binding site at the interface of two neighboringa1m monomers in the crystal is shown with the 2mFo-DFc electron densitycontoured at 0.8σ. The crystallographic twofold symmetry axis isindicated at the center. The distance between the two Ni²⁺ ions (cyan)is 7.8 Å, indicating independent complex formation (instead of abinuclear inorganic complex). Water molecules within 3.5 Å distance tothe metal ions as well as some crystallographically definedhydrogen-bonded water molecules in the second coordination shell areshown (red).

FIG. 2. A potential heme binding site in the three-dimensional structureof human α1m.

FIG. 2A depicts part of the loop region of a1m (orange) with asuperimposed structural segment of microsomal prostaglandin E synthase(PGES, pink; PDB ID: 2PBJ) including its bound heme group—using the Cαpositions of the common TCP[F/W] motif. In this model, side chainrotamers of His123 and Cys34 were so chosen that the relative axialposition to the central Fe³⁺ ion were optimized. For reasons of clarity,only PGES residues 102-120 are shown.

For comparison, in FIG. 2B, nitrophorin 4 from Rhodnius prolixus isdepicted as complex with its bound heme group within the central cavityand an axial ammonia ligand (PDB ID: 1X8P). Side chains within 4 Åaround the heme group are shown as sticks.

FIG. 3. Comparison of human a1m with three structurally most relatedlipocalins.

FIG. 3A depicts structure-based sequence alignment of human a1m (SEQ IDNO: 59) denoted as “a1m”) in comparison with human complement componentC8γ (PDB ID: 2QOS, chain C) (SEQ ID NO: 22, denoted as “C8γ”), humanL-prostaglandin D synthase (PDB ID: 3O2Y, chain B) (SEQ ID NO: 23,denoted as “PGDS”), and human lipocalin 15 (PDB ID: 2XST, chain A) (SEQID NO: 24, denoted as “Lcn15”). Expression tags were omitted andmutations in the recombinant proteins (a1m: Cys³⁴Ser; C8γ: Cys⁴⁰Ala,Asp⁹⁸Gly; PGDS: Cys⁶⁵Ala; Lcn15: Thr¹⁷Ser, Ala¹⁸Met) were reverted toreflect their natural amino acid sequences. Residues for which X-raycoordinates were missing are shaded grey. The characteristic Gly-X-Trpmotif of the lipocalins, the pair of Cys residues that gives rise to thetopologically conserved disulfide bridge, and the single unpaired Cysresidue are printed in bold. Insertions relative to a1m are indicated bylowercase letters. α-helices and β-strands were derived from therespective coordinates and are indicated by pink and green color,respectively. 100% conserved residues are labelled with stars. The 58structurally conserved residues in the β-barrel region of each lipocalin(Skerra, A. (2000) Biochim Biophys Acta 1482, 337-350), whose Cαpositions were used to align the four structures in panel (b), areboxed.

FIG. 3B shows the ribbon representation of the superimposed crystalstructures of a1m, C8γ, PGDS, and Lcn15 based on the 58 Cα positionsindicated in (a) and here depicted in darker shades of grey. Theresulting RMSD values versus a1m are 0.76 Å (C8γ), 1.02 Å (PGDS), and0.84 Å (Lcn15). Loops are colored individually (orange, a1m; palegreen,C8γ; marineblue, PGDS; pink, Lcn15). Two sequence stretches in the loopregion are not resolved in the Lcn15 structure.

FIG. 4. Model of the a1m/bikunin precursor protein (AMBP).

This structural model is based on the X-ray structure of a1m asdescribed herein (residues 27-190 in AMBP, orange) and the previouslypublished crystal structure of the serine protease inhibitor bikunin(residues 230-339 in AMBP, pink; PDB ID: 1BIK).

The linker region was modeled in a random conformation; the proteolyticcleavage site therein (residues 202-205) is indicated by an arrow. TheN-terminal 19 residue secretory signal peptide, which is processed bysignal peptidase, is omitted. The loop region around the ligand pocketof a1m is highlighted (cyan). The two internal repeat regions of bikunin(green) that are presumably involved in the inhibitory interaction withtarget serine proteases were identified by superposition with thetrypsin/trypsin inhibitor complex (PDB ID: 2PTC). Disulfide bridges areshown in a ball-and-stick representation.

FIGS. 5A-5B. Multiple alignment of 29 orthologous sequences of humanAMBP (UniProt ID: P02760) from various vertebrate species calculated byUniprot BLAST.

Sequences with a score lower than 350 as well as preliminary data wereomitted. Only residues 1-184 of the mature human a1m protein (firstline), corresponding to positions 20-202 of the translated human AMBPgene, were included. Taxonomic classes are labeled by colors: blue(mammals), red (amphibia), and green (fish). Secondary structureelements of a1m are indicated with pink (α-helices) and green(β-strands) symbols above its sequence. Residues with a degree ofconservation of more than 95% are highlighted in gray, except forGly23-Trp25 (cyan), Cys34 (orange), Cys72 (yellow) and Cys169 (yellow).The trivial names and the corresponding UniProt IDs (in parentheses)are: Gallus gallus: chicken (F1NUF8) (SEQ ID NO: 25), Meleagrisgallopavo: common turkey (G1MR91) (SEQ ID NO: 26), Pongo abelii:sumatran orangutan (Q5NVR3) (SEQ ID NO: 27), Nomascus leucogenys:northern white-cheeked gibbon (G1S4D1) (SEQ ID NO: 28), Callitrixjacchus: white-tufted-ear marmoset (F6R1P3) (SEQ ID NO: 29), Oryctolaguscuniculus: rabbit (G1TSY8) (SEQ ID NO: 30), Mus musculus: mouse (Q07456)(SEQ ID NO: 31), Rattus norvegicus: rat (Q64240) (SEQ ID NO: 32),Meriones unguiculatus: mongolian jird (Q62577) (SEQ ID NO: 33),Mesocricetus auratus: golden hamster (Q60559) (SEQ ID NO: 34), Bostaurus: bovine (P00978) (SEQ ID NO: 35), Sus scrofa: pig (P04366) (SEQID NO: 36), Ailuropoda melanoleuca: giant panda (G1M2K1) (SEQ ID NO:37), Felis catus: cat (E1CJT2) (SEQ ID NO: 38), Canis familiaris: dog(E2R796) (SEQ ID NO: 39), Equus caballus: horse (F6UZH0) (SEQ ID NO:40), Myotis lucifugus: little brown bat (G1PCS2) (SEQ ID NO: 41), Caviaporcellus: guinea pig (O70160) (SEQ ID NO: 42), Monodelphis domestica:gray short-tailed gray opossum (F7D6H6) (SEQ ID NO: 43), Xenopustropicalis: western clawed frog (Q6P2V8) (SEQ ID NO: 44), Xenopuslaevis: african clawed frog (P70004) (SEQ ID NO: 45), Ctenopharyngodonidella: Grass carp (A8VZJ0) (SEQ ID NO: 46), Danio rerio: zebrafish(A7E2Q2) (SEQ ID NO: 47), Oncorhynchus mykiss: rainbow trout (Q5F4T3)(SEQ ID NO: 48), Salmo salar: atlantic salmon (B5XD04) (SEQ ID NO: 49),Esox lucius: northern pike (C1BWU5) (SEQ ID NO: 50), Pleuronectesplatessa: european plaice (P36992) (SEQ ID NO: 51). Homo sapiens: humanis shown in SEQ ID NO: 1.

FIG. 6. A graphical illustration of the residues in a1m highlighted tobe considered for randomization.

-   a) Residues depicted in dark gray: a set of residues that    corresponds to the generic set of four structurally variable loops    at the entrance to the lipocalin ligand pocket (according to Skerra,    BBA 2000);-   b) Residues depicted in light gray: an additional set of “second    layer” residues whose side chains protrude into the ligand pocket    underneath the exposed loops.

FIG. 7. A model of a1m binding to Lu-DOTA-Bn conjugated to Biotin via aPEG4 linker.

FIG. 7A Illustration of simultaneous binding of Lu-DOTA-Bn-Linker-Biotin(black) by a1m (PDB code 3QKG) and a Streptavidin monomer (PDB code3RY2), that occurs during phage display selection. Linker length was acritical parameter to prevent sterical hindrance of a1m and Streptavidinduring simultaneous binding of the target.

FIG. 7B View into the cavity of a1m with a model of bound Lu-DOTA-Bn.The most prominent position of Lu-DOTA-Bn in the cavity revealed fromdocking experiments using the UCSF Chimera tool Chil2 was used for theconstruction of the a1m library. Amino acid positions chosen for randommutagenesis are indicated as sticks.

FIGS. 8A-8B. Representative non-nature ligands can be bound by a1mmuteins FIGS. 8A-8B show targets of Lu-DOTA-Bn-dPEG4-Biotin andColchicine-dPEG4-Biotin that were used for selection of a1m muteinsagainst the respective target.

FIG. 9. Biochemical characteristics of a1m muteins containing amino acidsubstitution due to insertion of first BstXI restriction site forlibrary cloning in comparison to wild-type human a1m.

FIG. 9A Coomassie-stained 15% SDS-PAGE analysis of whole cell extracts(1) before and (2) after induction of protein production for a1m andmuteins derived therefrom under reducing conditions. TG1/F− cellstransformed with the expression plasmid pa1m2 containing the indicatedamino acid substitution were grown in LB-Amp medium until an OD550 of0.5 was reached. The periplasmic protein production was with addition of0.2 μg/ml aTc (anhydrotetracycline) for 3 h, followed by periplasmicprotein extraction. M represents the molecular weight marker with thecorresponding band sizes in kDa displayed on the left of the gel. Bandsof an estimated molecular weight of 22 kDa corresponding to a1m muteinsare marked by an error.

FIG. 9B Size exclusion chromatography (SEC) profiles of a1m and muteinsderived therefrom. Proteins were subjected to Streptavidin-affinitychromatography (SAC) prior to SEC using PBS as running buffer and ananalytical grade Superdex 75 HR 10/30 column. Peak intensities differeddepending on the expression yield of the particular mutein.

FIG. 9C Coomassie-stained 15% SDS-PAGE analysis of soluble, monodispersea1m and muteins derived therefrom under reducing and non-reducingconditions after SEC.

FIG. 9D Comparison of protein yields after SAC and SEC of a1m andmuteins derived therefrom determined by measurement of absorbance at 280nm. Portion of aggregated protein after SEC was calculated in relationto 100% monomer. R20Q mutation increased expression level and monomer:aggregate ratio compared to wild-type human a1m.

FIG. 10. PCR assembly strategy for the simultaneous random mutagenesis

FIG. 10A illustrates the polymerase chain reaction (PCR) assemblystrategy for the simultaneous random mutagenesis of the 17 amino acidpositions 34, 36, 37, 47, 62, 64, 73, 75, 77, 90, 97, 99, 116, 118, 126,128, and 130 (underlined and numbered) in the amino acid sequence ofa1m. Whenever possible, codons optimized for E. coli expression wereused throughout for the non-mutated amino acid positions within theBstXI cassette since a1m gene was cloned from human AMBP gene(Swiss-Prot ID: AMBP_HUMAN; UniProt ID: P02760, corresponding to thehuman mature a1m sequence (without the valine residue at position 203 ofAMBP) (Meining & Skerra (2012) Biochem J. 445, 175-182). The 17positions were divided into four sequence subsets. For randomization ofthe amino acids in each subset an oligodeoxynucleotide was synthesized(SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5) wherein amixture of 19 different triplets i.e. all amino acids except cysteine(AAA, AAC, ACT, ATC, ATG, CAG, CAT, CCG, CGT, CTG, GAA, GAC, GCT, GGT,GTT, TAC, TCT, TGC, TGG, TTC) were employed at the mutated codons. Eachtrimer indicated by XXX encodes for a different amino acid except Cys.Four additional oligodeoxynucleotides (SEQ ID NO: 6, SEQ ID NO: 7, SEQID NO: 8 and SEQ ID NO: 9) with fixed nucleotide sequences correspondingto the non-coding strand (written below the DNA double strand sequencein 3′-5′ direction) and filling the gaps between the aforementionedoligodeoxynucleotides were also used in the assembly reaction. Twoflanking oligodeoxynucleotides (SEQ ID NO: 10 and SEQ ID NO: 11), whichwere added in excess and carried biotin groups at their 5′-end, servedas primers for the PCR amplification of the assembled, entirelysynthetic gene fragment. The two flanking primers each encompassed aBstXI restriction site, giving rise to mutually non-compatible overhangsupon enzyme digest. This special arrangement of restriction sitesenabled a particularly efficient ligation and cloning of the syntheticgene. Substitution of the amino acid Arg20 to Gln with respect to theoriginal a1m sequence was necessary to introduce the first BstXIrestriction site, while the insertion of the second one was possiblewithout altering the amino acid sequence. Additionally, amino acid Trp95was replaced by His for removal of a solvent-exposed, hydrophobicresidue, and His122 was substituted by Gln preventing complexion ofmetal-ion with the neighbouring His123. Furthermore, the unpairedresidue Cys34 was replaced by Ser in order to prevent unwanted disulfideformation. After one pot PCR the resulting gene fragment was insertedinto a vector providing the missing parts of the a1m structural gene.FIG. 10A discloses SEQ ID NOS 10, 2, 62, 64, 3, 60, 6, 4, 5, 61, 7, 8, 9and 11, respectively, in order of appearance.

FIG. 10B Design of the vector pNGAL108 for phage display selection ofa1m muteins. This plasmid is based on the generic E. coli expressionvector pASK75, which harbors for the tetracycline promoter/operator fortightly regulated transcriptional control. The tet^(o/o) is chemicallyinducible with anhydrotetracycline. The a1m expression cassetteencompasses an N-terminal OmpA signal peptide for periplasmic secretion,the mature part of the engineered lipocalin, the Strep-tag II foraffinity purification, followed by the gene III minor coat protein offilamentous bacteriophage M13 for phage display selection.

FIG. 11. An overlay of elution profiles revealed from phage displayselection after cycles 1-4 of a1m muteins specific for Lu-DOTA-Bn (FIG.11A) or Colchicine (FIG. 11B) starting from a synthetic combinatoriallibrary with 17 randomized positions.

Following an on-bead panning strategy, 100 nM of the biotinylated targetwas adsorbed onto Streptavidin- or NeutrAvidin-coated magnetic particlesprior to the incubation with about 10¹³ phagemids for 2 h in panningcycle 1 and for 1 h in panning cycles 2-4, respectively. After 10washing steps with PBS/0.1T containing 0.1 mM D-Desthiobiotin,specifically bound phagemids were eluted under competitive conditionusing 100 μM of unbiotinylated target. The phagemid titer of certainwash fractions and the elution fraction were determined and plottedsemi-logarithmically as fractional amount of totally applied phagemidsof each panning cycle (relative phagemid titer). The eluted phagemidswere then amplified and subjected to the next panning cycle.

FIG. 12. Results of the screening ELISA for identification ofLu-DOTA-Bn-specific a1m muteins after the fourth panning cycle.

FIG. 12A shows the experimental set-up for the ELISA. The BstXI cassetteof the enriched phagemid pool was subcloned into the expression plasmidpa1m2, which encodes a fusion of the OmpA signal peptide for theperiplasmic production in E. coli and the a1m coding region with theC-terminal Strep-tag II. Small-scale expression of randomly picked,single clones of a1m was performed in TG1/F⁻ overnight at 20° C.,followed by release of recombinant protein from the bacterial periplasmusing BBS (borate buffered saline) buffer supplemented with 1 mg/mllysozyme. The periplasmic extract was then applied to a 96-well Maxisorpplate, which was coated with 5 μg/ml Streptavidin and incubated with 0.5μM biotinylated Lu-DOTA-Bn. Target-bound muteins were detected using themurine anti Strep-tag-II monoclonal antibody Strep-MAB-Classic, which inturn was bound by an anti-mouse IgG (Fc-specific)/alkalinephosphatase-conjugate. Signal development upon addition of p-nitrophenylphosphate was followed by measuring the absorption at 405 nm in aSpectraMax 250 reader for up to 1.5 h.

FIG. 12B Histogram representing the measured absorptions at 405 nm forcertain a1m muteins. For explicit numbering of the analyzed clones, theprefix D1 was used to indicate them as Lu-DOTA-Bn-specific Anticalins,followed by the position in the 96-well master plate.

FIG. 12C Histogram representing the measured absorptions at 405 nm forcertain a1m muteins, which were normalized for the a1m signal. Forexplicit numbering of the analyzed clones, the prefix D1 was used toindicate them as Lu-DOTA-Bn-specific muteins, followed by the positionin the 96-well master plate.

FIG. 13. illustrates an amino acid sequence alignment of theLu-DOTA-Bn-specific a1m muteins D1A10 (SEQ ID NO: 12), D1A11 (SEQ ID NO:13), D1B1 (SEQ ID NO: 14, D1E1 (SEQ ID NO: 15), and D1H3 (SEQ ID NO: 16)with wild-type human a1m. Randomized positions are marked by an x. Lowercase letters indicate conserved amino acid substitutions to introduce apair of unique BstXI restrictions site (R20Q), to remove a tryptophaneresidue from the proteins surface (W95H) and to prevent metalcomplexation through by two histidine residues (H122Q). The eightstructurally conserved β-strands (A-H) and the four structurallyvariable loops (#1-4) are labeled. Dots represent amino acids identicalto the wild-type human a1m sequence (SEQ ID NO: 1). FIG. 13 disclosesthe “Library” sequence as SEQ ID NO: 65.

FIG. 14. Results of the screening ELISA for identification ofColchicine-specific a1m muteins after the fourth panning cycle.

FIG. 14A shows the experimental set-up for the ELISA. The BstXI cassetteof the enriched phagemid pool was subcloned into the expression plasmidpa1m2, which encodes a fusion of the OmpA signal peptide for theperiplasmic production in E. coli and the a1m coding region with theC-terminal Strep-tag II. Small-scale expression of randomly picked,single clones of a1m was performed in TG1/F⁻ overnight at 20° C.,followed by release of recombinant protein from the bacterial periplasmusing BBS (borate buffered saline) buffer supplemented with 1 mg/mllysozyme. The periplasmic extract was then applied to a 96-well Maxisorpplate, which was coated with 5 μg/ml Streptavidin and incubated with 0.5μM biotinylated Lu-DOTA-Bn. Target-bound muteins were detected using themurine anti Strep-tag-II monoclonal antibody Strep-MAB-Classic, which inturn was bound by an anti-mouse IgG (Fc-specific)/alkalinephosphatase-conjugate. Signal development upon addition of p-nitrophenylphosphate was followed by measuring the absorption at 405 nm in aSpectraMax 250 reader for up to 1.5 h.

FIG. 14B Histogram representing the measured absorptions at 405 nm forcertain a1m muteins. For explicit numbering of the analyzed clones, theprefix C1 was used to indicate them as Colchicine-specific Anticalins,followed by the position in the 96-well master plate.

FIG. 14C Histogram representing the measured absorptions at 405 nm forcertain a1m muteins, which were normalized for the a1m signal. Forexplicit numbering of the analyzed clones, the prefix C1 was used toindicate them as Colchicine-specific muteins, followed by the positionin the 96-well master plate.

FIG. 15. illustrates an amino acid sequence alignment of theColchicine-specific a1m muteins C1A1 (SEQ ID NO: 17), C1A2 (SEQ ID NO:18) and C1D1 (SEQ ID NO: 19) with wild-type human a1m. Randomizedpositions are marked by an x. Lower case letters indicate conservedamino acid substitutions to introduce a pair of unique BstXIrestrictions site (R20Q), to remove a tryptophane residue from theproteins surface (W95H) and to prevent metal complexation through by twohistidine residues (H122Q). The eight structurally conserved β-strands(A-H) and the four structurally variable loops (#1-4) are labeled. Dotsrepresent amino acids identical to the wild-type human a1m sequence (SEQID NO: 1). FIG. 15 discloses the “Library” sequence as SEQ ID NO: 66.

DETAILED DESCRIPTION

The present inventors have elucidated the secondary and tertiarystructure of human a1m lipocalin and potential target-binding sitesthereof. Accordingly, the present disclosure provides a collection ofamino acid sequence members comprised of muteins of a1m polypeptide or afunctional homologue thereof wherein the amino acid sequence of saidmembers differs from the a1m polypeptide at one or more of the sequencepositions of the wild type a1m polypeptide. These differences maycorrespond to the sequence positions in the four peptide loops #1, #2,#3 and #4 of human a1m polypeptide. The collection of amino acidsequence members can range in size from at least 10{circumflex over( )}2, at least 10{circumflex over ( )}3, at least 10{circumflex over( )}4, at least 10{circumflex over ( )}5, at least 10{circumflex over( )}6, at least 10{circumflex over ( )}7, at least 10{circumflex over( )}8, at least 10{circumflex over ( )}9, at least 10{circumflex over( )}10 and at least 10{circumflex over ( )}11.

The members of this collection have at least 60% sequence identity withhuman a1m. This includes all proteins that have a sequence homology oridentity of more than 60%, 70%, 80%, 85%, 90% or 95% in relation to thehuman mature a1m sequence (amino acid residues 20-203 of the translatedhuman AMBP gene (Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760; SEQ IDNO: 1).

In a preferred embodiment, at least one of said members can specificallybind to a target other than a target to which wild-type a1m binds andhas no or no substantial binding affinity for an endogenous a1m target.For example, an endogenous target of a1m can be retinoic acid orretinol. Preferably, the a1m lipocalin muteins disclosed herein do notspecifically bind retinoic acid or retinol in an assay as described inBreustedt, D. A., Schönfeld, D. L & Skerra, A. (2006) Comparativeligand-binding analysis of ten human lipocalins (Biochim. Biophys. Acta1764, 161-173). In a more preferred embodiment, at least one of saidmembers can specifically bind to two targets other than a target towhich wild-type a1m binds (e.g. Colchicine and Lutetium (177Lu)DOTA-TATE ((177)Lu-DOTA)); in this sense, a member contains two bindingsites. As used herein, a “target” is defined as any molecule to which ana1m mutein polypeptide of the disclosure is capable of specificallybinding, including all types of proteinaceous and non-proteinaciousmolecules such as haptens or other small molecules. As used herein, apolypeptide of the disclosure “specifically binds” a target if it isable to discriminate between that target and one or more referencetargets, since binding specificity is not an absolute, but a relativeproperty. “Specific binding” can be determined, for example, inaccordance with Western blots, ELISA, RIA-, ECL-, IRMA-tests, FACS, IHCand peptide scans. The polypeptide of the disclosure can bind to thetarget with an affinity in the micromolar or, in more preferredembodiments, in the nanomolar range. Binding constants of less than 100μM, 50 μM, 500 nM, 250 nM, 100 nM and 50 nM are also envisioned in thecurrent disclosure.

“Wild-type a1m”, when used herein, means the human mature a1m sequence(amino acid residues 20-203 of the translated human AMBP gene(Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760; SEQ ID NO: 1). Thewild-type a1m does preferably not contain a mutation in any one of theloops #1, #2, #3 and #4 as described herein. Whereas a “mutein of a1m”or “mutein a1m” or the like terms does contain at least one mutation inany one of the loops #1, #2, #3 and #4 as described herein in comparisonto wild-type a1m. Loop #1 comprises preferably amino acids 29-48, loop#2 comprises preferably amino acids 63-80, loop #3 comprises preferablyamino acids 89-100 and loop #4 comprises preferably amino acids 115-129of human mature a1m sequence. More preferably loop #1 comprises aminoacids 32-46 of mature a1m. More preferably loop #2 comprises amino acids66-72 of mature a1m. More preferably loop #3 comprises amino acids 91-98of human mature a1m sequence. More preferably loop #4 comprises aminoacids 118-126 of mature human mature a1m sequence.

The present disclosure also teaches a method of producing (i.e.isolating) a mutein of human a1m polypeptide or a functional homologuethereof, which specifically binds a target other than a target to whichwild-type a1m binds, comprising the steps of: (i) screening the membersof the collection with a target other than a target to which wild-typea1m binds in order to allow formation of a complex between said targetand at least one of said members, (ii) removing muteins having no or nosubstantial binding affinity for the target; and (iii) isolating themutein specifically binding to the target.

The disclosed crystal structure of human a1m polypeptide (see Example 1)serves as basis for the finding that a1m and a functional homologuethereof can provide suitable scaffolds for the generation ofpolypeptides having binding activity to a given target of interest. Theamino acid positions which are subjected to mutagenesis are distributedacross four sequence segments corresponding to four loops in thethree-dimensional structure of a1m. The number of the segments (loops)defined above which are used for mutagenesis can vary. It is notnecessary to mutate more than one of these four loops, for example, in aconcerted mutagenesis, but it is also possible to subject only one, twoor three of the loops to generate a mutein having detectable affinity toa given target of interest.

In one embodiment of the disclosed method, human a1m polypeptide may besubjected to mutagenesis at one of the sequence positions whichcorrespond to the linear polypeptide sequence positions 29-48, 63-80,89-100, and 115-129 of human a1m polypeptide. In a preferred embodimentof the disclosed method, human a1m polypeptide is subjected tomutagenesis at one of the sequence positions which correspond to thesequence positions 32-46, 66-72, 91-98, and 118-126 of human a1mpolypeptide. In a more preferred embodiment of the disclosed method,human a1m polypeptide is subjected to mutagenesis at one of the sequencepositions which correspond to the linear polypeptide sequence positions34-37, 62-64, 97-99, 116-118, and 126-130 of human a1m polypeptide.

In a still further embodiment of the disclosed method, the human a1mpolypeptide is subject to further mutagenesis at one of the sequencepositions which correspond to the linear polypeptide sequence positions30, 47, 73, 75, 77, 79 and 90 of human a1m polypeptide.

Accordingly, in one embodiment the disclosure provides an a1m muteinthat may contain a mutation at any one of the sequence positions whichcorrespond to the linear polypeptide sequence positions 29-48, 63-80,89-100, and 115-129 of human a1m polypeptide. In a preferred embodiment,an a1m mutein of the disclosure contains a mutation at any one of thesequence positions which correspond to the linear polypeptide sequencepositions 32-46, 66-72, 91-98, and 118-126 of human a1m polypeptide. Ina further embodiment, an a1m mutein of the disclosure contains amutation at one or more of the sequence positions which correspond tothe linear polypeptide sequence positions 34-37, 62-64, 97-99, 116-118,and 126-130 of human a1m polypeptide.

In a still further embodiment, the a1m mutein further contains amutation at one of the sequence positions which correspond to the linearpolypeptide sequence positions 30, 47, 73, 75, 77, 79 and 90 of humana1m polypeptide.

As provided in the current disclosure, an a1m mutein is preferably apolypeptide in which one or more amino acids within one, two, three, orall four loops are changed in comparison to a wild-type (or reference)a1m of the present disclosure (e.g. human a1m polypeptide). Said one ormore amino acids include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more amino acids that can be changed in loop#1, loop #2, loop #3 and/or loop #4.

However, it is also envisaged that, within loop #1 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 amino acids; within loop #2 1, 2, 3, 4,5, 6 or 7 amino acids; within loop #3 1, 2, 3, 4, 5, 6, 7 or 8 aminoacids; and/or within loop #4 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids canbe replaced.

Preferably, it is also envisaged that within loop #1 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids;within loop #2 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17or 18 amino acids; within loop #3 1, 2, 3, 4, 5, 6, 7, 8, 9; 10, 11 or12 amino acids; and/or within loop #4 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids can be replaced.

“Replacement”, when used herein, means that an amino acid different fromthat present at the corresponding position in the wild type a1mlipocalin is present in an a1m lipocalin mutein of the presentdisclosure.

The term “position”, when used in the present disclosure, means theposition of either an amino acid within an amino acid sequence depictedherein or the position of a nucleotide within a nucleic acid sequencedepicted herein. The term “corresponding” as used herein also includesthat a position is not only determined by the number of the precedingnucleotides/amino acids. Accordingly, the position of a given amino acidin accordance with the disclosure which may be substituted may vary dueto deletion or addition of amino acids elsewhere in a (mutant orwild-type) lipocalin. Similarly, the position of a given nucleotide inaccordance with the present disclosure which may be substituted may varydue to deletions or additional nucleotides elsewhere in a mutein or wildtype a1m 5′-untranslated region (UTR) including the promoter and/or anyother regulatory sequences or gene (including exons and introns).

Thus, under a “corresponding position” in accordance with the presentdisclosure it is preferably to be understood that nucleotides/aminoacids may differ in the indicated number but may still have similarneighbour ring nucleotides/amino acids. Said nucleotides/amino acidswhich may be exchanged, deleted or added are also comprised by the term“corresponding position”. When used herein “at a position correspondingto a position” a position in a “query” (or reference) amino acid (ornucleotide) sequence is meant that corresponds to a position in a“subject” amino acid (or nucleotide) sequence. A preferred query (orreference) sequence is shown in SEQ ID NO: 1.

Specifically, in order to determine whether a nucleotide residue oramino acid residue of the amino acid sequence of an a1m different froman a1m mutein of the disclosure corresponds to a certain position in thenucleotide sequence or the amino acid sequence of an a1m mutein asdescribed, a skilled artisan can use means and methods well-known in theart, e.g., alignments, either manually or by using computer programssuch as BLAST 2.0 (Altschul et al. (1990), J. Mol. Biol. 215:403-10),which stands for Basic Local Alignment Search Tool, or ClustalW(Thompson et al. (1994), Nucleic Acids Res. 22(22):4673-80) or any othersuitable program which is suitable to generate sequence alignments.Accordingly, wild-type a1m (SEQ ID NO: 1) can serve as “subjectsequence”, while the amino acid sequence of an a1m different from a1m asdescribed herein serves as “query sequence”.

Given the above, a skilled artisan is thus readily in a position todetermine which amino acid position mutated in a1m as described hereincorresponds to an amino acid of an a1m scaffold other than a1m.Specifically, a skilled artisan can align the amino acid sequence of amutein as described herein, in particular an a1m mutein of thedisclosure with the amino acid sequence of a different a1m to determinewhich amino acid(s) of said mutein correspond(s) to the respective aminoacid(s) of the amino acid sequence of said different lipocalin.

When used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid) or “mutant” refers to the exchange, deletion, or insertionof one or more nucleotides or amino acids, respectively, within the a1mof the present disclosure compared to the wild-type (naturallyoccurring) nucleic acid or protein “reference” scaffold of a1m, forexample, shown in SEQ ID NO: 1.

Accordingly, an a1m mutein of the present disclosure may include thewild type (natural) amino acid sequence of the “parental” proteinscaffold (human a1m) outside the mutated one or more amino acid sequencepositions within one, two, three or four loop(s); alternatively, an a1mmutein may also contain amino acid mutations outside the sequencepositions subjected to mutagenesis that do not interfere with thebinding activity and the folding of the mutein. Such mutations can beaccomplished on a DNA level using established standard methods. In apreferred embodiment, possible alterations of the amino acid sequenceare insertions or deletions as well as amino acid substitutions. Suchsubstitutions may be conservative, i.e. an amino acid residue isreplaced with a chemically similar amino acid residue. Examples ofconservative substitutions are the replacements among the members of thefollowing groups: 1) alanine, serine, and threonine; 2) aspartic acidand glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine;5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine,tyrosine, and tryptophan. One the other hand, it is also possible tointroduce non-conservative alterations in the amino acid sequence. Inaddition, instead of replacing single amino acid residues, it is alsopossible to either insert or delete one or more continuous amino acidsof the primary structure of a parental protein scaffold, where thesedeletions or insertion result in a stable folded/functional mutein,which can be readily tested by the skilled artisan.

Moreover, the skilled artisan will appreciate methods useful to prepareprotein muteins contemplated by the present disclosure but whose proteinor nucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence can include,e.g., directed mutagenesis of single amino acid positions in order tosimplify sub-cloning of a mutated lipocalin gene or its parts byincorporating cleavage sites for certain restriction enzymes. Inaddition, these mutations can also be incorporated to further improvethe affinity of a lipocalin mutein for a given target. Furthermore,mutations can be introduced to modulate certain characteristics of themutein such as to improve folding stability, serum stability, proteinresistance or water solubility or to reduce aggregation tendency, ifnecessary. For example, naturally occurring cysteine residues may bemutated to other amino acids to prevent disulphide bridge formation.

When used herein, “a1m”, in which one or more amino acid replacements,in particular at one or more position in one, two, three or all fourloops, are made in accordance with the teaching of the presentdisclosure, encompasses any other a1m known in the art or which can beidentified by using wide-type human a1m (SEQ ID NO: 1) as referencesequence, for example, in a BLAST search or using a nucleic acidmolecule encoding Blc as probe in, for example, a hybridizationexperiment.

Preferred a1m scaffolds other than wide-type human a1m, in which one ormore amino acid replacements, in particular at one or more position inone, two, three or all four loops, can be made in accordance with theteaching of the present disclosure, can, for example, be retrieved fromthe sequences shown in FIG. 5 (i.e. from a1m orthologues). Thus, an“a1m”, when used herein, may be an orthologue of wide-type human a1mshown in SEQ ID NO: 1.

The present disclosure also provides a crystal structure of a1m. In aparticular embodiment, the present disclosure includes the crystalstructure depicted in FIG. 1. The present disclosure also includes ana1m crystal being characterized by the data shown in Table 1.Preferably, the a1m crystal has space group P3₂21 and unit-cellparameters a=b=66.72, c=80.26, a=b=90.0, g=120.0.

TABLE 1 Data collection and refinement statistics Data collection Spacegroup P3₂21 Unit-cell parameters (Å, °) a = b = 66.72, c = 80.26, a = b= 90.0, g = 120.0 Resolution (Å) 46.89-2.17 Total reflections 114328Unique reflections 11272 Completeness (%)  96.7 (80.0)^(a) <I/s(I)> 20.2(1.1)^(a) Redundancy 10.4 (7.3)^(a) Mosaicity (°) 0.306 R_(merge) (%)  7.3 (169)^(a) Refinement^(b) R/R_(free) (%) 21.3/26.0 Protein residues164 Ligand molecules Ni²⁺, glycerol Solvent molecules 47 R.m.s.deviations from ideality: Bond lengths (Å) 0.017 Bond lengths (Å) 0.017Bond angles (°) 1.703 Average B values (Å²): Protein 71.8 Solvent 74.7Ramachandran plot outliers^(c): Most favoured region 130 Additionallyallowed region 13 Generously allowed region 1 Disallowed region 2^(a)Values in parentheses are for the highest resolution shell(2.29-2.17 Å). ^(b)For the last refinement cycle only data to 2.3 Åresolution were used. ^(c)Values by PROCHECK (Laskowski et al. (1993),J. Appl. Cryst. 26, 283-291).

The term “mutagenesis” as used herein means that the amino acidnaturally occurring at a sequence position of a1m can be substituted byat least one amino acid that is not present at this specific position inthe respective natural polypeptide sequence. The term “mutagenesis” alsoincludes modifying the length of sequence segments by deletion orinsertion of one or more amino acids. Thus, it is within the scope ofthe disclosure that, for example, one amino acid at a chosen sequenceposition is replaced by a stretch of three random mutations, leading toan insertion of two amino acid residues compared to the length of (therespective segment) of the wild-type protein. Such an insertion ofdeletion may be introduced independently from each other in any of thepeptide segments that can be subjected to mutagenesis in the disclosure.The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated into a selected sequenceposition during mutagenesis with a certain probability.

The term “collection” or “library” as used herein means that at leasttwo muteins that differ from each other in their amino acid sequencesare present. The upper limit of muteins generated by mutagenesis isusually restricted by the experimental conditions and is generallybetween 10⁷ and 10¹².

Such experimental conditions can, for example, be achieved byincorporating codons with a degenerate base composition in thestructural gene at those positions which are to be mutated. For example,use of the codon NNK or NNS (wherein N=adenine, guanine or cytosine orthymine; K=guanine or thymine; S=adenine or cytosine) allowsincorporation of all 20 amino acids plus the amber stop codon duringmutagenesis, whereas the codon VVS limits the number of possiblyincorporated amino acids to 12 since it excludes the amino acids Cys,Ile, Leu, Met, Phe, Trp, Tyr, Val from being incorporated into theselected position of the polypeptide sequence; use of the codon NMS, forexample, restricts the number of possible amino acids to 11 at aselected sequence position since it excludes the amino acids Arg, Cys,Gly, Ile, Leu, Met, Phe, Trp, Val from being incorporated at a selectedsequence position. In a preferred embodiment of the method of thedisclosure, a random mutagenesis is carried out, in which at least 4,preferably 6, more preferably 8 to 12 amino acids are allowed to beincorporated into a selected sequence position of a1m. In a particularlypreferred embodiment, at least one sequence position is subjected tocomplete randomization, i.e. all 20 amino acids are allowed to beincorporated at this position during mutagenesis. From the above, it isalso possible that the amino acid naturally present at a certainsequence position of a1m may also be present in the mutein after havingsubjecting this position to mutagenesis. It is also possible to use asdescribed by Wang, L., et al., Science, 292:498-500, 2001 or Wang, L.,Schultz, P. G., Chem. Comm., 1:1-11, 2002 “artificial” codons such asUAG which are usually recognized as stop codons in order to insert otherunusual amino acids, for example O-methyl-L-tyrosine orp-aminophenylalanine.

The term “α₁-Microglobulin” (“α1m” or “a1m”) as used herein is notlimited to the mature human α₁-Microglobulin (20-203 of the human AMBPgene (Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760; SEQ ID NO: 1), butincludes all polypeptides having the structurally conserved lipocalinfold and a sequence homology or identity with respect to the amino acidsequence of the human a1m (e.g. 20-202 of the human AMBP gene, ascomprised in the recombinant protein described in Example 1). Thisincludes all proteins that have a sequence homology or identity of morethan 60%, 70%, 80%, 85%, 90% or 95% in relation to the human a1m (20-202of the human AMBP gene (Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760);SEQ ID NO: 1). Preferably, such homologous sequences do not have amutation or replacement in loops #1. #2, #3 and #4 as described herein.

The term “lipocalin fold” is used in its regular meaning as used, e.g.,in Flower, D. R. (1996) or Skerra, A. (2000), supra, to describe thetypical three-dimensional lipocalin structure with a conformationallyconserved β-barrel as a central motif made of a cylindrically closedβ-sheet of eight antiparallel strands, wherein the open end of thebarrel the β-strands are connected by four loops in a pairwise manner sothat the binding pocket is formed (see also FIG. 1). A representativelipocalin fold is the human a1m fold.

The definition of the “peptide loops” or “loops” as used in the presentdisclosure is in accordance with the regular meaning of the human a1mfold, as also illustrated in Example 1 and FIG. 1.

The term “homology” as used herein has its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby a aspartate residue) at equivalent positions in the linear amino acidsequence of two proteins that are compared with each other, while theterm “sequence identity” refers to the number of amino acids that areidentical between two amino acid sequences at a particular amino acidposition. Percent identity is determined by dividing the number ofidentical residues by the total number of residues and multiplying theproduct by 100. The term “reference sequence” and “wild type sequence”(of a1m) is used interchangeably herein.

The percentage of homology can be determined herein using the programBLASTP, version blastp 2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al.(1997) Nucleic Acids Res. 25, 3389-3402). The percentage of homology isbased on the alignment of the entire polypeptide sequences (cutoff valueset to 10⁻³) including the propeptide sequences, using the human a1m asreference in a pairwise comparison. It is calculated as the percentageof numbers of “positives” (homologues amino acids) indicated as resultin the BLASTP program output divided by the total number of amino acidsselected by the program for the alignment. It is noted in thisconnection that this total number of selected amino acids can differfrom the length of the a1m (184 amino acids including the propeptide) asit is seen in the following.

The skilled artisan has in his disposal published sequence alignments oralignments methods. A sequence alignment can, for example, be carriedout as explained in WO 99/16873, using a published alignment such as theone in FIG. 1 of Redl, B. (2000) Biochim. Biophys. Acta, 1482, 241-248.If the three-dimensional structure of the lipocalins is availablestructural superpositions can also be used for the determination ofthose sequence positions that are to be subjected to mutagenesis in thepresent disclosure. Other methods of structural analysis such asmultidimensional nuclear magnetic resonance spectroscopy can also beemployed for this purpose.

The homologue of a1m can also be a mutein protein of a1m itself, inwhich amino acid substitutions are introduced at positions other thanthe positions selected in the present disclosure. For example, such amutein can be a protein in which positions at the solvent exposedsurface of the β-barrel are mutated compared to the wild type sequenceof the tear lipocalin in order to increase the solubility or thestability of the protein.

No or no substantial binding affinity means, under the used conditions,no complex is formed between the target and the collection of muteinswhich are contacted with the target. It is clear to the person skilledin the art that complex formation is dependent on many factors such asconcentration of the binding partners, concentration of compounds actingas competitors, ion strength of the buffers etc. The selection andenrichment is generally carried out under conditions which will allowisolation and enrichment of muteins having an affinity constant of atleast 10⁵ M⁻¹ to the target. However, the washing and elution steps canbe carried out under varying stringency. For example, if muteins havingan affinity constant of at least 10⁶ M⁻¹ are to be isolated, washing andelution can be performed under increased stringency, i.e. more stringentconditions. A selection with respect to the kinetic characteristics isalso possible. The selection can, for instance, be performed underconditions which favor complex formation of the target with muteins thatshow a slow dissociation from the target (receptor), or in other words alow k_(off) rate.

In a preferred embodiment of the disclosure, a nucleic acid coding forthe collection of muteins of the respective protein selected from a1m isused. In one embodiment, the nucleic acid results from mutagenesis andis operably fused with a gene coding for a polypeptide display moiety,such as the coat protein pIII of a filamentous bacteriophage of theM13-family or for a fragment thereof, in order to select at least onemutein for the binding of the given target. The fusion of a polypeptidedisplay moiety may be at the 5′ or 3′ end of the lipocalin mutein andpreferably is at the 3′ end.

The nucleic acid that results from mutagenesis can be accomplished by,e.g., PCR techniques. In a preferred embodiment of the method of thedisclosure, the generation of the nucleic acid coding for the mutatedsegments of the respective protein comprises the following two steps.First, two nucleic acid fragments, each of which codes for a part of themutated protein are generated by PCR such that these fragments arepartially overlapping. Second, these fragments are employed with twoflanking primers in order to obtain the nucleic acid comprising thecomplete mutated structural gene. Due to the overlap, the full-lengthPCR product can be amplified in the course of this reaction, withoutthat the addition of any additional nucleic acid is required. The twofragments, for example, can be obtained with a pair or pairs of suitableprimers in two separate amplification reactions.

In another preferred embodiment of the disclosure, a nucleic acidmolecules (e.g. DNA and RNA) comprising the nucleotide sequences codingone or more muteins as described herein are used. In this regard, itshould be note that since the degeneracy of the genetic code permitssubstitutions of certain codons by other codons specifying the sameamino acid, the disclosure is not limited to a specific nucleic acidmolecule encoding a disclosed mutein or fusion protein but includes allnucleic acid molecules comprising nucleotide sequences encoding afunctional mutein or fusion protein.

A nucleic acid molecule disclosed here may be “operably linked” to aregulatory sequence (or regulatory sequences) to allow expression ofthis nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or capable “to allow expression of anucleotide sequence” if it comprises sequence elements which containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions comprise a promoter which, inprokaryotes, contains both the promoter per se, i.e. DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the −35/−10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′-capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, preferably a promoter sequence. In anotherpreferred embodiment, a nucleic acid molecule of the disclosurecomprises a promoter sequence and a transcriptional terminationsequence. Suitable prokaryotic promoters are, for example, the tetpromoter, the lacUV5 promoter or the T7 promoter. Examples of promotersuseful for expression in eukaryotic cells are the SV40 promoter or theCMV promoter.

The nucleic acid molecules of the disclosure can also be comprised in avector or any other cloning vehicles, such as plasmids, phagemids,phage, baculovirus, cosmids or artificial chromosomes. In a preferredembodiment, the nucleic acid molecule is comprised in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage (e.g.M13K07, VCS-M13 or R408) intact phage particles are produced, therebyenabling physical coupling of the encoded heterologous cDNA to itscorresponding polypeptide displayed on the phage surface (reviewed,e.g., in Kay, B. K. et al. (1996) Phage Display of Peptides andProteins—A Laboratory Manual, 1st Ed., Academic Press, New York N.Y.;Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 401-424 orRodi, D. J. & Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinof the disclosure, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding lipocalin muteins of the disclosure, and inparticular a cloning vector containing the coding sequence of such alipocalin mutein can be transformed into a host cell capable ofexpressing the gene. Transformation can be performed using standardtechniques (Sambrook, J. et al. (1989), supra). Thus, the disclosure isalso directed to a host cell containing a nucleic acid molecule asdisclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of thedisclosure. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) orprimary mammalian cells.

The coding sequence for a1m, used as scaffold in the disclosure, canserve as a starting point for mutagenesis of the peptide segmentsselected by the person skilled in the art. The coding sequence of a1mhas been described by Breustedt, D. A., Schönfeld, D. L., and Skerra, A.(2006) Biochim Biophys Acta 1764, 161-173. For the mutagenesis of theamino acids in the four peptide loops, the person skilled in the art hasat his disposal the various known methods for site-directed mutagenesisor for mutagenesis by means of the polymerase chain reaction. Themutagenesis method can, for example, be characterized in that mixturesof synthetic oligodeoxynucleotides, which bear a degenerate basecomposition at the desired positions, can be used for introduction ofthe mutations. The use of nucleotide building blocks with reduced basepair specificity, as for example inosine, is also an option for theintroduction of mutations into the chosen sequence segment or amino acidpositions. The procedure for mutagenesis of target-binding sites issimplified as compared to antibodies, since for a1m only four instead ofsix sequence segments—corresponding to the four above mentioned peptideloops—have to be manipulated for this purpose. A further possibility isthe so-called triplet-mutagenesis. This method uses mixtures ofdifferent nucleotide triplets each of which codes for one amino acid forthe incorporation into the coding sequence.

One of the various applicable methods for the introduction of mutationsin the region of the four selected peptide loops of a1m disclosed hereis based on the use of four oligodeoxynucleotides, each of which ispartially derived from one of the four corresponding sequence segmentsto be mutated. In the production of these oligodeoxynucleotides, theperson skilled in the art can employ mixtures of nucleic acid buildingblocks for the synthesis of those nucleotide triplets which correspondto the amino acid positions to be mutated, so that codons or anticodonsrandomly arise for all amino acids or, according to the genetic code andto the composition of this mixture, for a selection of the desired aminoacids at this position.

For example, the first oligodeoxynucleotide corresponds in itssequence—apart from the mutated positions—at least partially to thecoding strand for the peptide loop, which is located in the polypeptidesequence of a1m at the most N-terminal position. Accordingly, the secondoligodeoxynucleotide corresponds at least partially to the non-codingstrand for the second sequence segment following in the polypeptidesequence. The third oligodeoxynucleotide corresponds in turn at leastpartially to the coding strand for the corresponding third sequencesegment. Finally, the fourth oligodeoxynucleotide corresponds at leastpartially to the non-coding strand for the fourth sequence segment. Apolymerase chain reaction can be performed with the respective first andsecond oligodeoxynucleotide and separately if needed, with therespective third and fourth oligodeoxynucleotide by using the nucleicacid which encodes the scaffold protein and/or its complementary strandas a template.

The amplification products of both of these reactions can be combined byvarious known methods into a nucleic acid which comprises the sequencefrom the first to the fourth sequence segments and which bears themutations at the selected amino acid positions. To this end, saidamplification products can, for example, be subjected to a newpolymerase chain reaction using flanking oligodeoxynucleotides asprimers as well as one or more mediator nucleic acid molecules whichcontribute the sequence between the second and the third sequencesegment. In the choice of the number of the oligodeoxynucleotides usedfor the mutagenesis and their arrangement within the gene sequence ofprotein used, the person skilled in the art has furthermore numerousalternatives at his disposal.

The nucleic acid molecules which code for the sequence regionencompassing the four peptide loops of a1m and which contain mutationsat the selected positions mentioned above can, for example, be connectedby ligation with the missing 5′- and 3′-sequences of a nucleic acidcoding for a1m and/or the vector disclosed below, and can be cloned in aknown host organism. A multitude of procedures are at the skilledperson's disposal for the ligation and the cloning. For example, in thecourse of an amplification, synthetic nucleic acid molecules withrestriction endonuclease recognition sequences, which are also presentat the corresponding positions in the nucleic acid sequence for a1m, canbe attached at both ends of the nucleic acid to be cloned so that aligation is made possible following hydrolysis with the correspondingrestriction enzyme. The missing 5′- and 3′-sequences of a nucleic acidcoding for a1m can also be attached to the nucleic acid moleculecomprising the mutated sequence positions via PCR.

Longer sequence segments within the gene coding for the protein selectedfor mutagenesis can also be subjected to random mutagenesis via knownmethods, for example, by use of the polymerase chain reaction underconditions of increased error rate, by chemical mutagenesis or by usingbacterial mutator strains (Low et al., J. Mol. Biol. 260 (1996),359-368). Such methods can also be used for the further optimization ofthe target affinity or target specificity of a mutein which has alreadybeen produced. Mutations which possibly occur outside the segments ofthe sequence positions 32-46, 66-72, 91-98, and 118-126 of a1m, forinstance, can often be tolerated or can even prove advantageous, forexample if they contribute to an improved folding efficiency or foldingstability of the mutein.

After having brought the coding nucleic acid sequences that weresubjected to mutagenesis to expression, the clones carrying the geneticinformation for the collection of respective muteins which bind a giventarget can be selected from the library obtained. Known expressionstrategies and selection strategies can be employed for the selection ofthese clones. Methods of this kind have been described in the context ofthe production or the engineering of recombinant antibody fragments,such as the “phage display” technique (Hoess, Curr. Opin. Struct. Biol.3 (1993), 572-579; Wells and Lowman, Curr. Opin. Struct. Biol. 2 (1992),597-604) or “colony screening” methods (Skerra et al., Anal. Biochem.196 (1991), 151-155) or “ribosome display” (Roberts, Curr. Opin. Chem.Biol. 3 (1999) 268-273).

An embodiment of the “phage display” technique (Hoess, supra; Wells andLowman, supra; Kay et al., Phage Display of Peptides and Proteins—ALaboratory Manual (1996), Academic Press) is given here as an example ofa selection method according to the disclosure for muteins with thedesired binding characteristics. For the exemplary selection method,phasmids are produced which effect the expression of the mutated a1mstructural gene as a fusion protein with a signal sequence at theN-terminus, preferably the OmpA-signal sequence, and with the coatprotein pIII of the phage M13 (Model and Russel, in “TheBacteriophages”, Vol. 2 (1988), Plenum Press, New York, 375-456) orfragments of this coat protein, which are incorporated into the phagecoat, at the C-terminus. The C-terminal fragment ΔpIII of the phage coatprotein, which contains only amino acids 217 to 406 of the natural coatprotein pIII, is preferably used to produce the fusion proteins.Especially preferred is a C-terminal fragment from pIII in which thecysteine residue at position 201 is missing or is replaced by anotheramino acid. The various other possible embodiments of the “phagedisplay” technique are at disposal of person skilled in the art.

The fusion protein can contain other components, for example, anaffinity tag or an epitope sequence for an antibody which allows theimmobilization or the later purification of the fusion protein or itsparts. Furthermore, a stop codon can be located between the regioncoding for a1m or its mutein and the gene segment for the coat proteinor its fragment, which stop codon, preferably an amber stop codon, is atleast partially translated into an amino acid during translation in asuitable suppressor strain.

Phasmids here denote plasmids which carry the intergenetic region of afilamentous bacterial phage, such as for example M13 or f1 (Beck andZink, Gene 16 (1981), 35-58) or a functional part thereof, so thatduring superinfection of the bacterial cells with a helper phage, forexample M13K07, VCS-M13 or R408, one strand of the circular phasmid DNAis packaged with coat proteins and is exported into the medium asso-called phagemid. On the one hand this phagemid has the a1m muteinencoded by the respective phasmid built into its surface as a fusionwith the coat protein pIII or its fragment, wherein the signal sequenceof the fusion protein is normally cleaved off. On the other hand itcarries one or more copies of the native coat protein pIII from thehelper phage and is thus capable of infecting a recipient generally abacterial strain carrying an F- or F′-plasmid. In this way a physicalcoupling is ensured between the packaged nucleic acid carrying thegenetic information for the respective a1m mutein, and the encodedprotein which is at least partially presented in functional form on thesurface of the phagemid.

A vector can, for example, be used in the construction of the phasmidwith the sequences coding for the a1m muteins. The nucleic acid codingfor the peptide loops can, for example, be inserted into the vector viaboth of the BstXI-restriction sites. Recombinant phasmids areincorporated by transformation into the E. coli strain, for exampleXL1-blue (Bullock et al., BioTechniques 5 (1987), 376-379) or TG1. Inthis way, clones are made which can produce many different a1m muteinsas fusion proteins.

The disclosed library, i.e. the collection of the a1m muteins obtainedby the taught methods, is subsequently superinfected in liquid cultureaccording to known methods with an M13-helper phage. After thisinfection the incubation temperature of the culture can be reduced forproduction of the phagemids. Preferred incubation temperatures are thosein which the optimal folding of the a1m mutein as a component of thefusion protein with the phage coat protein or its fragment is expected.During or after the infection phase the expression of the gene for thefusion protein with the a1m mutein can be induced in the bacterialcells, for example by addition of anhydrotetracycline. The inductionconditions are so chosen that a substantial fraction of the phagemidsproduced presents at least one a1m mutein. The phagemids are isolatedafter a culture incubation phase of for example 6 to 8 hours. Variousmethods are known for isolation of the phagemids, such as for exampleprecipitation with polyethylene glycol.

The isolated phasmids can be subjected to a selection by incubation withthe desired target, wherein the target is present in a form allowing atleast a temporary immobilization of those phagemids carrying muteinswith the desired binding activity as fusion proteins in their coat.Among the various embodiments known to the person skilled in the art,the target can for example be conjugated with a carrier protein such asserum albumin and be bound via this carrier protein to a protein bindingsurface, for example polystyrene. Microtiter plates suitable for ELISAtechniques or so-called “immuno-sticks” can preferably be used for thisimmobilization of the target. Alternatively, conjugates of the targetcan also be implemented with other binding groups such as for examplebiotin. The target can then be immobilized on surfaces which selectivelybind this group, such as for example microtiter plates or paramagneticparticles coated with streptavidin or avidin.

Residual protein or phagemid-binding sites present on the surfaces whichare charged with targets can be saturated with blocking solutions knownfor ELISA-methods. The phagemids are for example subsequently brought incontact in a physiological buffer with the target immobilized on thesurface. Unbound phagemids are removed by multiple washings. Thephagemid particles remaining on the surface are subsequently eluted. Forelution, the free target can be added as a solution. But the phagemidscan also be eluted by addition of proteases or, for example, in thepresence of acids, bases, detergents or chaotropic salts, or undermoderately denaturing conditions. A preferred method is the elutionusing buffers of pH 2.2, wherein the eluate is subsequently neutralized.

Afterwards, E. coli cells are infected with the eluted phagemids usinggenerally known methods. The nucleic acids can also be extracted fromthe eluted phagemids and be incorporated into the cells in anothermanner. Starting from the E. coli clones obtained in this way, phagemidsare in turn generated by superinfection with M13-helper phages accordingto the method described above and the phagemids propagated in this wayare once again subjected to a selection on the surface with theimmobilized target. Multiple selection cycles are often necessary inorder to obtain the phagemids with the muteins of the disclosure inenriched form. The number of selection cycles is preferably chosen suchthat in the subsequent functional analysis at least 0.1% of the clonesstudied produce muteins with detectable affinity for the given target.Depending on the size, i.e. the complexity of the library employed, 2 to8 cycles are typically required to this end.

For the functional analysis of the selected muteins, an E. coli straincan be infected with the phagemids obtained from the selection cyclesand the corresponding double stranded phasmid DNA is isolated. Startingfrom this phasmid DNA or also from the single-stranded DNA extractedfrom the phagemids, the nucleic acid sequences of the selected muteinsof the disclosure can be determined by the methods common for thispurpose and the amino acid sequence can be derived therefrom. Themutated region or the sequence of the entire a1m mutein can be subclonedin another expression vector and expressed in a suitable host organism.The vector mentioned above can, for example, be used as the expressionvector and the expression can be performed in E. coli strains, forexample, E. coli-TG1. The muteins of a1m produced by genetic engineeringcan be purified by various proteinchemical methods. The a1m muteins soproduced (for example with said vector) carry the affinity peptideStrep-Tag II (Schmidt et al., supra) at their C-terminus and, therefore,can preferably be purified by streptavidin affinity chromatography.

The selection can also be carried out by means of other methods, forexample using “ribosome display” or “yeast (surface) display”, amongmany other methods. Many corresponding embodiments are known to theperson skilled in the art or are described in the literature. Acombination of methods can also be applied. For example, clones selectedor at least enriched by “phage display” can additionally be subjected toa “colony screening”. This procedure has the advantage that individualclones can directly be isolated with respect to the production of an a1mmutein with detectable binding affinity for a non-natural target.

In addition to the use of E. coli as host organism in the “phagedisplay” technique or the “colony screening” method, other bacterialstrains, for example yeast or also insect cells or mammalian cells, canfor example be used for this purpose. In addition to the selection of ana1m mutein from a primary library produced starting from a codingnucleic acid sequence for a mutein, comparable methods can also beapplied in order to optimize a mutein with respect to the affinity orspecificity for the desired target by repeated, optionally limitedmutagenesis of its coding nucleic acid sequence.

It is additionally possible to subject the muteins produced as taught toa further, optionally partial random mutagenesis in order to selectvariants of even higher affinity from the new library thus obtained. Acorresponding procedures have already been described for the case ofdigoxigenin binding muteins of the bilin-binding protein for the purposeof an “affinity maturation” (DE 199 26 068, WO 00/75308; Schlehuber etal., supra) and can also be applied to a mutein disclosed here in acorresponding manner by the person skilled in the art.

The present disclosure also relates to a lipocalin mutein derived from apolypeptide of a1m or a functional homologue thereof, wherein the muteincomprises at least one mutated amino acid residues at any sequenceposition in the four peptide loops #1, #2, #3 and #4 (dark gray areas ofFIG. 6), wherein said a1m or functional homologue thereof has at least60% sequence homology with human a1m, and wherein the mutein binds agiven target with detectable affinity. This includes all proteins thathave a sequence homology or identity of more than 60%, 70%, 80%, 85%,90% or 95% in relation to the human a1m (20-203 of the human AMBP gene(Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760; SEQ ID NO: 1).

The muteins of the disclosure can have the natural amino acid sequenceof a1m outside the mutated segments, i.e. the regions of the amino acidpositions 20 to 30, 48 to 63, 80 to 89, 100 to 115 and 129 to 165 ofa1m. On the other hand, the muteins disclosed here can also containamino acid mutations outside the positions subjected to mutagenesiscompared to the wild-type a1m protein as long as those mutations do notinterfere with the binding activity and the folding of the mutein. Thisincludes that, for example, mutations, substitutions, deletions,insertion of amino acid residues as well as N- and/or C-terminaladditions can be introduced into the natural amino acid sequence of a1m.

Such modifications of the amino acid sequence of the selected proteinwithin or without the selected binding region include directedmutagenesis of single amino acid positions, for example, in order tosimplify the subcloning of the mutated lipocalin gene or its parts byincorporating cleavage sites for certain restriction enzymes. Forexample, mutations can be introduced into the a1m gene in order tosimplify the cloning of the mutated gene segment via two new BstXIrestriction sites at these positions. Furthermore, mutations can beintroduced within or without the four peptide loops in order to improvecertain characteristics of the mutein of the protein chosen as scaffold,for example its folding stability or folding efficiency or itsresistance to proteases.

In a preferred embodiment, for instance, Cys34 of a1m is exchanged toSer or Ala, whereby its covalent crosslinking with other proteins suchas immunoglobulin A (which might occur in in vivo applications of amutein) can be prevented and its monomeric structure can be stabilized.Similarly, Cys residues which may occur as a result of the mutagenesisare not always crucial for the binding of the given target and may besubstituted by Ala or other amino acids in order to prevent covalentbond formation or oxidation of the thiol group.

In a preferred embodiment, Cys34 can be substituted and/or the muteincarries one or more of the amino acid substitution compared to a1m. Inthis respect, it should be noted that the present disclosure is alsodirected to a (recombinant) a1m having the natural amino acid sequencesin which only Cys34 has been substituted for any other suitable aminoacid. This a1m polypeptide can be produced using the methods describedhere for the production of the other muteins of the disclosures, forexample by use of the vector disclosed below.

The disclosure also provides a monomeric lipocalin mutein that, due tothe potential to engineer two binding sites into the binding pocket ofthe mutein, can have binding specificity for two given ligands. Forvarious applications it could also be advantageous to have more than onebinding site per molecule available.

The one or more muteins of the disclosure may bind the desired targetwith a detectable affinity, i.e. with an affinity constant of preferablyat least 10⁵ M⁻¹. Affinities lower than this are generally no longermeasurable with common methods such as ELISA and are therefore ofsecondary importance for practical applications. Especially preferredare muteins which bind the desired target with an affinity of at least10⁶ M⁻¹, corresponding to a dissociation constant for the complex of 1μM. The binding affinity of a mutein to the desired target can bemeasured by the person skilled in the art by a multitude of methods, forexample by fluorescence titration, by competition ELISA or by thetechnique of surface plasmon resonance.

The target which is bound by the mutein can be any chemical moiety that,for example, can also be recognized and bound by an antibody(immunoglobulin). Accordingly, the target can be a chemical compound infree or conjugated form which exhibits features of an immunologicalhapten, a hormone such as steroid hormones or any biopolymer or fragmentthereof, for example, a peptide, a protein or protein domain, a peptide,an oligodeoxynucleotide, a nucleic acid, oligo- and polysaccharides oranother macromolecule or conjugates thereof. In a preferred embodimentof the disclosure, the target is a protein. The protein can be providedeither in free or conjugated form or as a fusion protein for theselection of muteins. In a further preferred embodiment of thedisclosure, the target is a hapten.

A1m itself shows binding activity for some endogenous chemicalcompounds. For example, free heme (e.g. from hemolysis) is known to havesevere destructive effects on proteins and DNA and can damage vitalorgans including the central nervous system (Kumar, S., andBandyopadhyay, U. (2005) Toxicol Lett 157, 175-188). Recently, a1m hasbeen shown to counteract the toxic effect of heme and also of otheroxidants, notably hydrogen peroxide and hydroxyl radicals, and to exertreductase activity (Olsson, M. G., Olofsson, T., Tapper, H., andÅkerström, B. (2008) Free Radic Res 42, 725-736; Allhorn, M., Berggärd,T., Nordberg, J., Olsson, M. L., and Åkerström, B. (2002) Blood 99,1894-1901; Allhorn, M., Klapyta, A., and Åkerström, B. (2005) Free RadicBiol Med 38, 557-567; Larsson, J., Allhorn, M., and Kerstrom, B. (2004)Arch Biochem Biophys 432, 196-204; Allhorn, M., Lundqvist, K.,Schmidtchen, A., and Åkerström, B. (2003) J Invest Dermatol 121,640-646). The identification of a potential heme binding site by thedisclosure provides a structural explanation for these physiologicalactivities of a1m. The putative association of the heme group with Cys34and the close contacts with Lys92, Lys118 and Lys130 are in agreementwith experimental observations that mutation or chemical blocking ofthese residues inhibits or at least decreases the reduces activity(Allhorn, M., Klapyta, A., and Åkerström, B. (2005) Free Radic Biol Med38, 557-567). Furthermore, a1m can act as a scavenger of free hemeitself and thereby help to remove it from sensitive tissues and tofinally excrete it via the kidney.

It is clear to the skilled person that complex formation is dependent onmany factors such as concentration of the binding partners, the presenceof competitors, ionic strength of the buffer system etc. Selection andenrichment is generally performed under conditions allowing theisolation of lipocalin muteins having an affinity constant of at least10⁵ M⁻¹ to the target. However, the washing and elution steps can becarried out under varying stringency. A selection with respect to thekinetic characteristics is possible as well. For example, the selectioncan be performed under conditions, which favor complex formation of thetarget with muteins that show a slow dissociation from the target, or inother words a low k_(off) rate.

An a1m mutein of the disclosure may be used for complex formation with anon-natural target. The target may be any chemical compound in free orconjugated form which exhibits features of an immunological hapten, ahormone such as steroid hormones or any biopolymer or fragment thereof,for example, a protein or protein domain, a peptide, anoligodeoxynucleotide, a nucleic acid, an oligo- or polysaccharide orconjugates thereof. In a preferred embodiment of the disclosure thetarget is a protein. The protein can be any globular soluble protein ora receptor protein, for example, a trans-membrane protein involved incell signaling, a component of the immune systems such as an MHCmolecule or cell surface receptor that is indicative of a specificdisease. The mutein may also be able to bind only fragments of aprotein. For example, a mutein can bind to a domain of a cell surfacereceptor, when it is part of the receptor anchored in the cell membraneas well as to the same domain in solution, if this domain can beproduced as a soluble protein as well. However the disclosure is by nomeans limited to muteins that only bind such macromolecular targets. Butit is also possible to obtain muteins of tear lipocalin by means ofmutagenesis which show specific binding affinity to ligands of low(er)molecular weight such as biotin, fluorescein or digoxigenin.

For some applications, it is useful to employ the muteins of thedisclosure in a labeled form. Accordingly, the disclosure is alsodirected to lipocalin muteins which are conjugated to a label selectedfrom the group consisting of enzyme labels, radioactive labels, coloredlabels, fluorescent labels, chromogenic labels, luminescent labels,haptens, digoxigenin, biotin, metal complexes, metals, and colloidalgold. The mutein may also be conjugated to an organic molecule. The term“organic molecule” as used herein preferably denotes an organic moleculecomprising at least two carbon atoms, but preferably not more than sevenrotatable carbon bonds, having a molecular weight in the range between100 and 2000 Dalton, preferably 1000 Dalton, and optionally includingone or two metal atoms.

In general, it is possible to label the lipocalin mutein with anyappropriate chemical substance or enzyme, which directly or indirectlygenerates a detectable compound or signal in a chemical, physical orenzymatic reaction. An example for a physical reaction is the emissionof fluorescence upon irradiation or the emission of X-rays when using aradioactive label. Alkaline phosphatase, horseradish peroxidase andβ-galactosidase are examples of enzyme labels which catalyze theformation of chromogenic reaction products. In general, all labelscommonly used for antibodies (except those exclusively used with thesugar moiety in the Fc part of immunoglobulins) can also be used forconjugation to the muteins of the present disclosure. Such conjugatescan be produced by methods well known in the art.

Affinity tags such as the Strep-tag or Strep-tag II (Schmidt, T. G. M.et al. (1996) J. Mol. Biol. 255, 753-766), the myc-tag, the FLAG-tag,the His₆-tag (SEQ ID NO: 52) or the HA-tag or proteins such asglutathione-S-transferase also allow easy detection and/or purificationof recombinant proteins are further examples of preferred fusionpartners. Finally, proteins with chromogenic or fluorescent propertiessuch as the green fluorescent protein (GFP) or the yellow fluorescentprotein (YFP) are suitable fusion partners for a lipocalin mutein of thedisclosure as well.

The present disclosure also relates to nucleic acid molecules (DNA andRNA) comprising nucleotide sequences coding for one or more muteins asdescribed herein. Since the degeneracy of the genetic code permitssubstitutions of certain codons by other codons specifying the sameamino acid, the disclosure is not limited to a specific nucleic acidmolecule encoding a fusion protein of the disclosure but includes allnucleic acid molecules comprising nucleotide sequences encoding afunctional fusion protein.

In addition, in one embodiment of the disclosure, the ligand that can bebound by a mutein derived from human a1m polypeptide or a functionalhomologue may be Colchicine, Lutetium (177Lu) DOTA-TATE ((177)Lu-DOTA)or fragments thereof. These ligands are derived from the plant Colchicumautumnale and a rare earth bound to the chelating ligand DOTA,respectively. But other targets may also be of mouse, rat, porcine,equine, canine, feline or bovine or cynomolgus origin, to name only afew illustrative examples. Targets may be so-called small moleculetargets, peptides, proteins, cellular moieties, to name only a fewillustrative examples.

Therefore, one aspect of the present disclosure is directed to a muteinderived from human a1m polypeptide or a functional homologue thereofthat contains a mutation at any one of the sequence positions whichcorrespond to the linear polypeptide sequence positions 32-46, 66-72,91-98, and 118-126 of human a1m polypeptide, and binds to Colchicine. Insome further embodiments, the a1m mutein contains a mutation at one ormore of the sequence positions which correspond to the linearpolypeptide sequence positions 34-37, 62-64, 97-99, 116-118, and 126-130of human a1m polypeptide. In a still further embodiment, the a1m muteinfurther contains a mutation at one of the sequence positions whichcorrespond to the linear polypeptide sequence positions 30, 47, 73, 75,77, 79 and 90 of human a1m polypeptide. Examples of a1m lipocalinmuteins that bind Colchicine are shown in SEQ ID NOs: 17, 18 and 19. Thepresent application also encompasses lipocalin muteins that bindColchicine and have a sequence homology or identity of 60% or more, suchas 70%, 80%, 85% or 90%, in relation to the a1m lipocalin mutein shownin SEQ ID NO: 17, 18, or 19, respectively. In some preferredembodiments, such lipocalin muteins have at least one loop that isidentical to a loop of as the a1m lipocalin mutein shown in SEQ ID NO:17, 18, or 19, respectively.

Moreover, yet another aspect of the present disclosure is directed to amutein derived from human a1m polypeptide or a functional homologuethereof that contains a mutation at any one of the sequence positionswhich correspond to the linear polypeptide sequence positions 32-46,66-72, 91-98, and 118-126 of human a1m polypeptide, and binds to(177)Lu-DOTA. In some further embodiments, the a1m mutein contains amutation at one or more of the sequence positions which correspond tothe linear polypeptide sequence positions 34-37, 62-64, 97-99, 116-118,and 126-130 of human a1m polypeptide. In a still further embodiment, thea1m mutein further contains a mutation at one of the sequence positionswhich correspond to the linear polypeptide sequence positions 30, 47,73, 75, 77, 79 and 90 of human a1m polypeptide. Examples of a1mlipocalin muteins that bind (177)Lu-DOTA are shown in SEQ ID NOs: 12,13, 14, 15 and 16. The present application also encompasses lipocalinmuteins that bind (177)Lu-DOTA and have a sequence homology or identityof 60% or more, such as 70%, 80%, 85% or 90%, in relation to any one ofthe a1m lipocalin muteins shown in SEQ ID NO: 12, 13, 14, 15 and 16. Insome preferred embodiments, such lipocalin muteins have at least oneloop that is identical to a loop of the a1m lipocalin mutein shown inSEQ ID NO: 12, 13, 14, 15, or 16, respectively.

Colchicine is an alkaloid derived from Colchicum autumnale possessinganti-inflammatory and antimitotic characteristics. Colchicine inhibitsmicrotubule polymerization by binding to tubulin, one of the mainconstituents of microtubules. Availability of tubulin is essential tomitosis, and therefore colchicine effectively functions as a mitoticpoison. The inhibiting function of colchicine has been of great use inthe study of cellular genetics. Apart from inhibiting mitosis,colchicine also inhibits neutrophil motility and activity, leading to anet anti-inflammatory effect. Colchicine is used for the treatment andprevention of gout as well as liver and primary biliary cirrhosis (ChenY-J. et al, (2008), Int. J. Phar. 350, 230-239). It has a narrowtherapeutic index and the potential for severe or fatal toxicity. Acutecolchicine poisoning can be caused intentionally, unintentionallybecause of non-optimal treatment or by mix up of Colchicum autumnalewith the nontoxic eatable wood garlic. In any case acute colchicinepoisoning is associated with a high mortality rate (Baud F. J., (1995),The New Engl. J. of Med., 642-643). An a1m mutein against colchicinecould act as antidote to treat colchicine poisoning.

Lutetium (Lu) is an element of the lanthanide series and thus belongs tothe rare earths, whereas DOTA is a chelating ligand binding transitionmetals and rare earths with high stability under physiologicalconditions (Corneillie, T. M. et al (2003), J. Am. Chem. Soc. 125,3436-3437). The radioactive form 177-Lu in complex with DOTA to form177-Lu-DOTA is a suitable tracer for radiotherapy of tumors and imagingas it emits both beta particles suitable for radiotherapy and gamma rayssuitable for imaging (Garske, U. et al (2012), Theranostics. 2,459-471). An a1m mutein binding to 117-Lu-DOTA combined with anothermutein binding to e.g. a target expressed on tumors can be used todirect the radionucletide 177-Lu to the tumor for therapy as well as forimaging.

As used herein, the singular forms “a”, “an”, and “the”, include pluralreferences unless the context clearly indicates otherwise. Thus, forexample, reference to “a lipocalin mutein” includes one or morelipocalin muteins.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure described herein. Such equivalents areintended to be encompassed by the present disclosure.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or” as used herein.

Several references and documents are cited throughout the text of thisspecification. Each of the documents cited herein (including allpatents, patent applications, scientific publications, SWISS-PROT DataBank Accession Numbers, Swiss-Prot IDs, UniProt IDs, etc.), whethersupra or infra, are hereby incorporated by reference in their entirety.Nothing herein is to be construed as an admission that the disclosure isnot entitled to antedate such disclosure by virtue of prior disclosure.

The following non-limiting Examples and Figures further illustratevarious aspects of the present disclosure.

EXAMPLES Example 1 Structural Analysis of Human a1m

To provide insight into the suitability and properties of human a1m forgeneration of lipocalin muteins having binding affinity to a giventarget, the present disclosure teaches the three-dimensional structureof the unglycosylated human a1m. For crystal preparation and X-raystructural analysis the DNA sequence encoding amino acids 20-202 of thehuman AMBP gene (Swiss-Prot ID: AMBP_HUMAN, UniProt ID: P02760) wasexpressed with an N-terminal OmpA signal peptide and a C-terminalStrep-tag II from the plasmid pASK75-a1m2-strep as previously described(Breustedt, D. A., Schönfeld, D. L & Skerra, A. (2006) Comparativeligand-binding analysis of ten human lipocalins. Biochim. Biophys. Acta1764, 161-173.), while Cys34 was replaced by Ser in order to preventundesired disulfide bond formation.

The fold of a1m is characterized by a central eight-stranded calyx-likeβ-barrel, which is open to the solvent at one end, as well as a shortα-helical stretch between strands A and B (loop #1, at the open end) anda C-terminal long α-helix following strand H which tightly packs againstthe β-strands G, H and A on the outside of the β-barrel (FIG. 1). Thus,a1m shows the typical structural features of a kernel lipocalin (Flower,D. R. (1996) Biochem J 318, 1-14; Skerra, A. (2000) Biochim Biophys Acta1482, 337-350) wherein four structurally variable loops (designated as#1, #2, #3 and #4), which connect pairs of neighboring β-strands at theopen end of the calyx, form the entrance to a large cavity.

Loop #1 and the β-strands B, C, and D, with crystallographic B-factorsof 64-131 Å², appear considerably more flexible than the β-strands E, F,G, H, and A (B-factors: 38-65 Å²) and also the loop #4. Regions ofhighest structural flexibility comprise the short α-helix within loop #1as well as its connection to β-strand B (Pro35-Met44), loop #2 betweenβ-strands C and D (Trp67-Cys72) and the loop connecting β-strands B andC (Glu53-Glu59) at the closed bottom of the β-barrel. As a consequence,side chains of residues in these regions were partly difficult to model.

Residues Cys72 and Cys169 form a disulfide bridge that links thestructurally defined C-terminal residues of a1m to the transition regionbetween loop #2 and β-strand D and represents a conserved structuralfeature within the lipocalin family (Skerra, A. (2000) Biochim BiophysActa 1482, 337-350). Residues Cys34, Lys92, Lys118 and Lys130 werepreviously found to be involved in the association with chromophores(Berggärd, T., Cohen, A., Persson, P., Lindqvist, A., Cedervall, T.,Silow, M., Thogersen, I. B., Jonsson, J. A., Enghild, J. J., andÅkerström, B. (1999) Protein Sci 8, 2611-2620; Kwasek, A., Osmark, P.,Allhorn, M., Lindqvist, A., Åkerström, B., and Wasylewski, Z. (2007)Protein Expr Purif 53, 145-152). In this study, the single unpairedCys34 had been mutated to Ser and was thus not available for chromophorebinding. This residue is positioned in the transition region betweenstrand A and loop #1 and is therefore freely accessible to the solvent,thus in principle allowing linkage to IgA or other binding partners inhuman plasma. The two N-glycosylation sites at Asn17 and Asn96 (Ekström,B., Lundblad, A., and Svensson, S. (1981) Eur J Biochem 114, 663-666;Escribano, J., Lopex-Otin, C., Hjerpe, A., Grubb, A., and Mendez, E.(1990) FEBS Lett 266, 167-170; Amoresano, A., Minchiotti, L., Cosulich,M. E., Campagnoli, M., Pucci, P., Andolfo, A., Gianazza, E., andGalliano, M. (2000) Eur J Biochem 267, 2105-2112) are also accessible tosolvent and should sterically allow sugar attachment.

Lys118 and Lys92 are located at the upper rim of the central cavitywhile Lys130 is positioned half way down towards its bottom. Lys118 andLys130 are within a 9 Å distance from Ser(Cys)34 and point into thebinding site, thus enabling a ligand to interact with all three sidechains. Their spatial relationship with Lys92, which is 13 Å away fromLys118, is less obvious. Notably, two other residues, Met62 and Met99,which both point into the cavity, could be involved in chromophorebinding, as well. However, neither of them is absolutely conserved amongorthologous a1m species (cf. FIG. 5). Nevertheless, except for a fewcrystallographic water molecules around the side chains of Lys92,Lys118, and Lys130, there was no indication for additional electrondensity close to any of these residues and, hence, the ligand pocket inthe crystallized protein appears rather empty.

The central cavity of a1m is about 13 Å deep with an approximatediameter of around 13 Å at its top and 7 Å at its bottom (FIG. 1b ).With these dimensions, the calyx is wider than in most other lipocalins.The electrostatic potential map exhibits a distinct positive patterninside the pocket as well as in the region of the four loops at itsopening while negative charge prevails at the outer surface of thislipocalin, in particular around the closed end of the

-barrel (cf. FIG. 1b ). Eight basic residues (Arg43, Arg66, Arg68,Lys69, Lys92, Lys94, Lys118, Lys130), notably without acidiccounterpart, line the upper part of the pocket, whereas the lower cavityis shaped by hydrophobic side chains. There, several aromatic residues(Tyr79, Phe88, Phe114, Tyr132) are available for π-stacking and provideinteractions for ligand binding. The pronounced positive potentialinside the calyx together with the hydrophobic environment towards itsbottom indicate a preference for negatively charged ligands with ahydrophobic moiety.

The corresponding structure-based multiple sequence alignment of a1mwith the sequences of the three lipocalins (human complement componentC8γ (Lovelace, L. L., Chiswell, B., Slade, D. J., Sodetz, J. M., andLebioda, L. (2008) Mol Immunol 45, 750-756) (PDB ID: 2QOS, chain C),human L-prostaglandin D synthase (PGDS, unpublished; PDB ID: 302Y, chainB; for the closely related mouse ortholog, PDB ID: 2CZU, see Kumasaka,T., Aritake, K., Ago, H., Irikura, D., Tsurumura, T., Yamamoto, M.,Miyano, M., Urade, Y., and Hayaishi, O. (2009) J Biol Chem 284,22344-22352), and human lipocalin 15 (Lcn15, unpublished, PDB ID: 2XST)reveals that relative positions and lengths of the secondary structureelements largely coincide, whereas there are only minor sequence gapsbetween strands B/C, E/F and G/H, corresponding to the loop segments(FIG. 3a ). Notably, as a typical feature of the lipocalins, despitehigh similarity in their fold the mutual sequence similarity is verylow: of 184 residues in total, a1m shares only 40 (22%) identicalresidues with C8γ, 41 (22%) residues with PGDS, and merely 30 (16%)residues with Lcn15. Among those, 15 residues are common to all fourlipocalin sequences (cf. FIG. 3a ), including positions within thepreviously described structurally conserved regions (SCRs) and, inparticular, the characteristic lipocalin signature Gly²³-Xaa-Trp²⁵(Flower, D. R. (1996) Biochem J 318, 1-14). Structural superposition ofthe four lipocalins indicates that the β-barrel core structure is highlyconserved, whereas the loops #1, #2, #3 and #4 considerably vary inconformation (FIG. 3b ).

A Cys residue equivalent to Cys34 of a1m is also present in the naturalsequences of Lcn15 (UniProt ID: Q6UWW0) and C8γ (UniProt ID: P07360).Other human plasma lipocalins carry a similar free thiol group, whichseems to generally provide for intermolecular cross-linking, atdifferent positions. Lcn2/NGAL (UniProt ID: P80188), for example,carries such a Cys residue at position 87, which is equivalent to Asp83in a1m. PGDS (UniProt ID: P41222), on the other hand, exhibits acatalytically active Cys residue inside the cavity at position 65, whichwould be equivalent to Ser47 in a1m.

In order to compare the sequence variation among closely relatedorthologous a1m species from several vertebrates, a BLAST search ofresidues 20-202 of the translated human AMBP gene against the UniProtKBsequence database was performed and 28 unique sequences with a score ofhigher than 350 were aligned (FIG. 5). In this alignment 36 residues of183 in total are conserved to at least 95%. Six blocks of residues withelevated levels of conservation can be discerned: four blocks(Gln13-Phe16, Gly23-Trp25, Thr106-Tyr111 and Leu13-Arg134) are locatedaround the calyx bottom, one block (Gly167-Pro171) harbors Cys169involved in the lipocalin-typical disulfide bridge with the fullyconserved residue Cys72, and one block (Thr33-Trp36) encompasses threeresidues of the putative heme binding site.

Notably, none of the residues that were previously postulated to beresponsible for chromophore binding (Cys34, Lys92, Lys118, and Lys130)is conserved among the paralogous lipocalins mentioned above, while(except for Lys92) the same residues are highly conserved amongorthologous a1m sequences. Also, the four-residue heme-binding sequenceTCPW is highly conserved among the orthologs but diverse in otherlipocalins (cf. FIG. 3a ). His123, which is involved in the Ni²⁺ bindingsite, is absolutely conserved only among a1m from mammals while thecorresponding sequences from amphibia mostly exhibit Gly or Ser at thisposition. The first N-glycosylation site, Asn17, is not conservedwhereas the second one at Asn96 is only conserved among mammals.

Example 2 A Nickel Binding Site at the Interface Between TwoCrystallographic Neighbor Monomers

At the tip of loop #4, the symmetry-related side chains His122 andHis123′ from two neighboring protein molecules in the crystal latticecoordinate a metal ion, together with four water molecules. The electrondensity of the metal has a pronounced peak height of 12.6 σ, and basedon the presence of a high NiCl₂ concentration in the crystallizationsolution and on the apparent coordination geometry (Rulisek, L., andVondrasek, J. (1998) Journal of inorganic biochemistry 71, 115-127) itwas assigned as a Ni²⁺ ion. Due to the local crystallographic C2symmetry, there is a second equivalently bound Ni²⁺ ion at a closedistance of 7.8 Å, coordinated by His122′ and His123 (FIG. 1c ).

The ligand environment of the metal ion shows almost perfect octahedralgeometry. The distances of His-N to the Ni²⁺-ion are 1.9 Å and 2.3 Å,respectively, and the distances of waters are in the range of 2.2-2.3 Å,except for one water molecule (W4) that appears more tightly bound at1.7 Å. The observed distances correspond well to the average distancesof 2.18 Å for Ni—N and 2.28 Å for Ni—OH₂ that were deduced from mediumresolution structures of proteins carrying Ni²⁺-binding sites (Zheng,H., Chruszcz, M., Lasota, P., Lebioda, L., and Minor, W. (2008) J InorgBiochem 102, 1765-1776) and also to the corresponding average valuesfrom the MESPEUS database (Ni—N: 2.19 Å) and (Ni—OH₂: 2.31 Å) (Hsin, K.,Sheng, Y., Harding, M. M., Taylor, P., and Walkinshaw, M. D. (2008)Journal of Applied Crystallography 41, 963-968). Three further watermolecules at a distance of 2.4-2.6 Å to the Ni²⁺-coordinating waters,one of them shared between the two metal centers, are part of a secondhydration shell. Lys118-NZ, Lys118-O, Ser120-OG, Arg121-N, andThr126-OG1 are all in hydrogen bonding distances to at least one ofthese water molecules. The double Ni²⁺-binding site explains the clearlysupportive effect of NiCl₂ during crystallization of α₁m.

The nickel binding site arises between two adjacent crystallographicmonomers as part of an interface with a total contact surface of 690 Å².The complex significance score (CSS) of this interface as calculated byPISA is low with a value of 0.154. The protein dimer interface isfurther stabilized by π-stacking between Trp36 and Arg121′ of theadjacent monomer as well as minor interactions between residues 66-69,73-75, 90-99, and 118-123. As there is no biochemical evidence for acondition under which α₁m would preferentially form a dimer in solution,this interface is most likely a crystal packing artifact. Theunglycosylated α₁m monomer is the predominant form at saltconcentrations of 100-200 mM NaCl, and the presence of 1 mM NiSO₄ (or 1mM EDTA) did not indicate metal-dependent protein dimerization duringsize exclusion chromatography.

Example 3 Human α1m Contains a Potential Binding Site that May Allow forthe Interaction with a Ligand (Endogenous Target)

Human α₁m is known to bind retinoic acid and retinol, two endogenousphysiological compounds, with dissociation constants around 1 μM(Breustedt, D. A., Schönfeld, D. L & Skerra, A. (2006) Comparativeligand-binding analysis of ten human lipocalins. Biochim. Biophys. Acta1764, 161-173.). In addition, there are multiple indications frombiochemical experiments for the ability of α₁m to bind and interact withheme (Allhorn, M., Berggärd, T., Nordberg, J., Olsson, M. L., andÅkerström, B. (2002) Blood 99, 1894-1901; Larsson, J., Allhorn, M., andKerstrom, B. (2004) Arch Biochem Biophys 432, 196-204; Allhorn, M.,Lundqvist, K., Schmidtchen, A., and Åkerström, B. (2003) J InvestDermatol 121, 640-646), yet so far without a structural basis.Surprisingly for the first time, the present disclose teaches α₁mcrystal structure for the presence of a typical heme binding site.

In a recent study, the CXXCH, FXXGXXCXG (SEQ ID NO: 53) and CP sequencesignatures were identified as common heme binding motifs in proteins(Li, T., Bonkovsky, H. L., and Guo, J. T. (2011) BMC Struct Biol 11,13). Indeed, the solvent accessible Cys34-Pro35 dipeptide that occurs inloop #1 appears as a possible site of interaction with heme. Proteinswith known 3D structures in which CP dipeptides are involved in hemebinding are the enzymes CYP121 from M. tuberculosis (Leys, D., Mowat, C.G., McLean, K. J., Richmond, A., Chapman, S. K., Walkinshaw, M. D., andMunro, A. W. (2003) J Biol Chem 278, 5141-5147) (PDB ID: 1N40) andprostacyclin 12 synthase (PGIS) from zebrafish (Li, Y. C., Chiang, C.W., Yeh, H. C., Hsu, P. Y., Whitby, F. G., Wang, L. H., and Chan, N. L.(2008) J Biol Chem 283, 2917-2926) (PDB ID: 3B98), both belonging to thecytochrome P450 family, as well as the human nuclear receptorREV-ERBbeta (Pardee, K. I., Xu, X., Reinking, J., Schuetz, A., Dong, A.,Liu, S., Zhang, R., Tiefenbach, J., Lajoie, G., Plotnikov, A. N.,Botchkarev, A., Krause, H. M., and Edwards, A. (2009) PLoS Biol 7, e43)(PDB ID: 3CQV), chloroperoxidase (CPO) from C. fumago (Kuhnel, K.,Blankenfeldt, W., Terner, J., and Schlichting, I. (2006) J Biol Chem281, 23990-23998) (PDB ID: 2CIW) and the microsomal prostaglandin Esynthase (PGES) from M. fascicularis (Yamada, T., and Takusagawa, F.(2007) Biochemistry 46, 8414-8424) (PDB ID: 2PBJ).

Inspection of the region Thr33-Ser(Cys)34-Pro35-Trp36 in the crystalstructure of α₁m, which lies opposite to His123 in loop #4, reveals astriking similarity with the portion Thr109-Cys110-Pro111-Phe112 (SEQ IDNO: 54) of the heme binding site in PGES (FIG. 2). Indeed, superpositionof the Cα-atoms of the TS(C)PW tetrapeptide in recombinant α₁m with theones of the TCPF motif (SEQ ID NO: 54) in PGES results in anextraordinarily close match with an RMSD of 0.18 Å. Although thesequences of the corresponding motifs are less similar in the otherenzymes mentioned above, superposition with the tetrapeptidesLeu420-Cys421-Pro422-Gly423 (SEQ ID NO: 55) in PGIS (RMSD: 0.23 Å),Phe344-Cys345-Pro346-Gly347 (SEQ ID NO: 56) in CYP121 (RMSD: 0.12 Å),Val383-Cys384-Pro385-Met386 (SEQ ID NO: 57) in REV-ERBbeta (RMSD: 0.17Å), and Pro28-Cys29-Pro30-Ala31 (SEQ ID NO: 58) in CPO (RMSD: 0.17 Å)indicates a conserved conformation.

In PGES, the axial Fe³⁺ ligand position is occupied by the sulfhydrylgroup of the cosubstrate glutathione (GSH) while the Cys110-SH groupfrom the enzyme's active site is positioned on the same side of the hemegroup at a distance of 5.3 Å to the metal center. In contrast to PGES,the corresponding Cys residues in the enzymes CYP121, PGIS, CPO andREV-ERBbeta act as axial ligands of the iron ion. If the heme group werebound to α₁m in a similar orientation with respect to the CP motif as itappears in PGES, His123 in loop #4 would be able to act as the oppositeaxial ligand to the central heme iron (FIG. 2a ).

The close resemblance with PGES suggests that the putative heme bindingsite of α₁m may even allow for the interaction with an additionalbinding partner, for example GSH, next to the heme group. The cleftbetween loop #3 and loop #4 in α₁m seems sufficiently large and flexibleto accommodate both a heme group and another ligand or substrate, whichcould dive further into the deeper part of the lipocalin pocket. Theoverall positive potential of the cavity should assist in thestabilization of the negatively charged heme group and both Lys118 andHis122 would be able to form salt bridges with its propionatesubstituents. The almost absolutely conserved residues Trp36 and Trp95among α₁m orthologs (cf. FIG. 5) are both accessible to solvent in theapo-protein and, indeed, can promote association with lipophilic ligandssuch as heme by involving aromatic interactions.

TCP[FW] motifs with similar geometry can be found in a number of otherproteins not associated with heme. Most of these are either DNA bindingproteins (PDB IDs: 2AIK, 1GPC, 3C25, 1KB6, 3BVQ, 2ATQ) or enzymesinvolved in redox processes utilizing GSH as (co)substrate (PDB IDs:2HZF, 1KTE, 3GN3, 1ZH9, 2PBJ). In all cases where this motif has beenstructurally characterized it was shown that GSH can act as ligand. Thesuperposition of the redox enzyme TCP[FW] motifs with the correspondingtetrapeptide in α1m results in RMSD values between 0.08 Å (2HZF) and0.26 Å, while the TCP[FW] motifs of DNA binding proteins, with theexception of 2ATQ, align with higher RMSD values between 0.14 Å and 0.35Å. The remarkably close conformational match of the TCP[FW] motif amongthose proteins suggests that it constitutes a general structural featureassociated with GSH interaction. These findings imply that a1m might notonly be able to bind heme but also to accept GSH as a co-substrate orligand.

Example 4 Model of the α₁m/Bikunin Precursor as an Example of a NaturalBifunctional Fusion Protein

α₁m shares a common biological source with the Kunitz-type serineproteinase inhibitor bikunin as both originate from the so-calledα₁m/bikunin precursor protein (AMBP). Bikunin (Pugia, M. J., Valdes, R.,Jr., and Jortani, S. A. (2007) Adv Clin Chem 44, 223-245) is also knownas urinary trypsin inhibitor or inter-α-trypsin inhibitor light chain,which further encompasses the mast cell protease inhibitor trypstatin(Itoh, H., Ide, H., Ishikawa, N., and Nawa, Y. (1994) J Biol Chem 269,3818-3822). AMBP (UniProt ID: P02760) is expressed mainly in humanhepatocytes from the AMBP gene with a 19 residue leader sequence and,probably after signal peptide processing, becomes posttranslationallycleaved by a furin-like protease into the two mature proteins, which arethen separately glycosylated and finally excreted into the plasma(Åkerström, B., Lögdberg, L., Berggärd, T., Osmark, P., and Lindqvist,A. (2000) Biochim Biophys Acta 1482, 172-184; Tyagi, S., Salier, J. P.,and Lal, S. K. (2002) Arch Biochem Biophys 399, 66-72). The α₁m/bikuninprecursor (AMBP) was modeled by combining the structure of α₁m solvedhere with the previously published X-ray structure of bikunin (Xu, Y.,Carr, P. D., Guss, J. M., and Ollis, D. L. (1998) J Mol Biol 276,955-966) (FIG. 4), thus revealing a natural example of a trulybifunctional fusion protein.

Example 5 Primary Residues to be Considered for Randomization

As illustrated in FIG. 6, residues depicted in dark gray: 32-46, 66-72,91-98, 118-126, and residues depicted in light gray: 30, 47, 64, 73, 75,77, 79, 90, 99, 116, 128, are primary residues to be considered forrandomization in order to subsequently select (a) mutein(s) derived froma1m or a functional homologue thereof that can bind a target other thanan endogenous or natural target to which the wild-type a1m binds andwherein the mutein may have no or no substantial binding affinity forsuch endogenous or natural a1m target. Note that the reliable assignmentof these residues became only possible after our structural elucidationof α₁m.

Example 6 Preparation of Biotinylated Lu-DOTA-Bn

p-NH₂-Bn-DOTA (44.5 μmol; Macrocyclics Inc., Dallas, Tex., USA),18-Biotinamino-17-oxo-4,7,10,13-tetraoxa-16-azaicosan-1-oic acidsuccinimidyl ester (44.5 μmol; Iris Biotech GmbH, Marktredwitz,Germany), and triethylamine (26 μl, 2.8 mmol) were dissolved in 2 ml ofdry N,N-dimethylformamide. The reaction mixture was stirred for 16 h atroom temperature. The solvent was removed at vacuum. The remaining solidwas dissolved in water/acetonitrile 1:1 and purified by reversed-phaseHPLC (column: Merck Purospher Star RP-8e 250×10 mm; gradient 20-30%acetonitrile in water+0.1% (w/v) trifluoroacetic acid, tR=11.4 min). TheDOTA-biotin conjugate was obtained as a colourless solid (isolated: 29.5mg, 20.9 μmol, 47%, salt of trifluoroacetate) and subjected to ESI-MSanalysis: calculated [M+H]+=1068.5282, [M+2H]2+=534.7677,[M−H]=1066.5136; found [M+H]+=1068.5388, [M+2H]2+=534.7754,[M−H]−=1066.5332

Example 7 Preparation of Biotinylated Colchicine

Deacetylation of colchicine was carried out in three steps following thepublication of Bagnato et al. (Bagnato et al. (2004) J. Org. Chem. 69,8987-8996). Colchicine (0.50 mmol), 4-(dimethylamino)pyridine (0.50mmol), and triethylamine (141 μl, 1.0 mmol) were dissolved in 5 ml ofdry acetonitrile. After addition of di-tert-butyl dicarbonate (1.25mmol), the mixture was stirred under reflux. After 1 h additionaldi-tert-butyl dicarbonate (1.25 mmol) was added and refluxing wascontinued for another 2 h. After cooling to room temperature 25 ml ofdichloromethane were added and the solution was washed three times with25 ml of saturated aqueous citric acid. The combined aqueous solutionswere extracted once with 25 ml of dichloromethane. The organic layerswere combined and washed once with 25 ml of a saturated solution ofsodium chloride, dried with sodium sulfate, and evaporated to dryness.The resulting brownish solid was used without further purification.

The crude N-Boc-colchicine from the previous step was dissolved in 5 mlof methanol and treated with 4 ml of a 0.5 M solution of sodiummethoxide in methanol. The reaction was stirred for 90 min at roomtemperature, after which the reaction was quenched by the addition of120 mg (2.24 mmol) of ammonium chloride. The solvent was removed underreduced pressure. The crude product was purified by silicachromatography, using ethyl acetate/acetone 4:1 as eluent. The desiredproduct was obtained as a pale yellow solid (166 mg, 0.38 mmol, 75% over2 steps). Analytical data were in agreement with published data.

N-Boc-N-deacetylcolchicine (0.34 mmol) was dissolved in 4 ml ofdichloromethane. 400 μl of trifluoroacetic acid were added and themixture was stirred for 3 h at room temperature. The solvent was removedunder reduced pressure. After purification by silica chromatography(dichloromethane/methanol 9:1), the product was obtained as a yellowsolid (142 mg, 0.30 mmol, 9%, salt of trifluoroacetate). Analytical datawere in agreement with published data.

N-deacetylcolchicine (61 μmol of the trifluoroacetate),18-Biotinamino-17-oxo-4,7,10,13-tetraoxa-16-azaicosan-1-oic acidsuccinimidyl ester (55 μmol) and triethylamine (14 μl, 100 μmol) weredissolved in 2 ml of dry dichloromethane. The reaction mixture wasstirred for 24 h at room temperature. The solvent was removed underreduced pressure and the crude product was purified by silicachromatography (dichloromethane/methanol 9:1 to 7:1). The desiredcolchicine-biotin conjugate was obtained as a slightly yellowish solid(42 mg, 46 μmol, 84%) and subsequently applied to ESI-mass spectrometry:calculated [M+H]⁺=916.4373, [M−H]⁻=914.4226; measured [M+H]⁺=916.4358,[M−H]⁻=914.4219.

Example 8 Construction of a Mutant a1m Phage Display Library

Polymerase chain reaction (PCR) assembly of the a1m BstXI cassette asillustrated in FIG. 7 was essentially performed according to a publishedstrategy (Gebauer, Skerra (2012) Methods Enzymol 503, 157-188) in a onepot amplification reaction with oligodeoxynucleotides (SEQ ID NO: 2-11).Oligodeoxynucleotides were designed such that the primers with SEQ IDNO: 2-5 corresponded to the coding strand and carried one of 19different trimers at the amino acid positions 34, 36, 37, 47, 62, 64,73, 75, 77, 90, 97, 99, 116, 118, 126, 128, and 130 respectively, whileprimers with SEQ ID NO: 5-8 corresponded to the non-coding strand. Thetwo flanking primers with SEQ ID NO: 10 and SEQ ID NO: 11 were used inexcess and served for the amplification of the assembled randomized genefragment. In total, 15 PCR cycles using Taq DNA polymerase (Fermentas,St. Leon-Roth, Germany) were performed.

Oligodeoxynucleotides SEQ ID NO: 2-9 were synthesized using a ExpediteNucleic Acid Synthesize System (AME Bioscience, Bedfordshire, UK) andfurther purified by urea PAGE. Reaction tubes, solutions and nucleosidephosphoramidites were purchased from Sigma-Aldrich (SAFC ProligoReagents, Steinheim, Germany), whereas trimer phosphoramidites werepurchased from Glen Research (Sterling, Va., USA). The two flankingprimers with SEQ ID NO: 10 and SEQ ID NO: 11 were purchased in HPLCgrade from Thermo Fisher Scientific (Ulm, Germany). The resulting DNAlibrary was cut with BstXI (New England Biolabs, Schwalbach, Germany)and cloned on the phagemid vector phNGAL108 (SEQ ID NO:20), which isbased on the generic expression vector pASK75 (Skerra (1994) Gene 151,131-135), codes for a fusion protein composed of the OmpA signalpeptide, the synthetic a1m gene, the Strep-tag II followed by an ambercodon, and the full length gene III coat protein of the filamentousbacteriophage M13 (Vogt and Skerra (2004) Chem Bio Chem 5, 191-199).After electroporation of E. coli XL1-Blue (Bullock et al. (1987)Biotechniques 5, 376-378) with the ligation mixture of 3 μg digested PCRproduct and 30 μg digested plasmid DNA, 7.9×10⁹ transformants wereobtained.

After electroporation cells which were transformed with the phasmidvectors on the basis of phNGAL108, coding for the library of thelipocalin muteins as phage pIII fusion proteins were plated onto LB-Ampplates (Ø 15 cm) and incubated overnight at 37° C. Then, cells werescratched off the plates, diluted in 2YT medium to an OD550 of 0.1 withthe corresponding antibiotic added and cultured at 37° C. and 160 rpmuntil an OD550 of 0.6 was reached. After infection with VCS-M13 helperphage (Agilent Technologies, La Jolla, USA) at a multiplicity ofinfection of approximately 10 the culture was shaken for additional 30min at 37° C., 100 rpm. Then, kanamycin (70 μg/ml) was added to theculture, while lowering the incubator temperature to 26° C. andincreasing the shaker speed to 140 rpm. After 10 min gene expression wasinduced via addition of anhydrotetracycline (ACROS Organics, Geel,Belgium) at 25 μg/l (125 μl of a 200 μg/ml stock solution indimethylformamide, DMF per liter of culture). Incubation continued foranother 7 h at 26° C., 140 rpm.

Cells from the complete culture were sedimented by centrifugation (30min, 12,100 g, 4° C.). The supernatant containing the phagemid particleswas sterile-filtered (0.45 μm), mixed with ¼ volume 20% (w/v) PEG 8000,15% (w/v) NaCl, and incubated on ice for at least 2 h. Aftercentrifugation (60 min, 18,000 g, 4° C.), the supernatant was discardedand the sediment was recentrifugated (10 min, 18,000 g, 4° C.) tocompletely remove residual PEG solution. The precipitated phagemidparticles from 1 liter of culture were dissolved in 40 ml of cold,sterile BBS/E (200 mM Na-borate, 160 mM NaCl, 1 mM EDTA pH 8.0)containing 50 mM benzamidine (Sigma). The solution was incubated on icefor 30 min. After centrifugation of undissolved components (10 min,40,000 g, 4° C.) each supernatant was transferred to a new reactionvessel.

Addition of ¼ volume 20% w/v PEG 8000, 15% (w/v) NaCl and incubation for60 min on ice served to reprecipitate the phagemid particles until thephagemids were aliquoted and frozen at −80° C. for storage. For thefirst selection cycle phagemids were thawed and centrifuged (20 min,18,500 g, 4° C.), the supernatant was removed, and the precipitatedphagemid particles were dissolved and combined in a total of 400 μl PBScontaining 50 mM benzamidine. After incubation for 30 min. on ice, thesolution was centrifuged (5 min, 18,500 g, 4° C.) in order to removeresidual aggregates, and the supernatant was used directly for the phagedisplay selection.

Example 9 Selection of a1m Muteins with Affinity to Lu-DOTA-Bn by PhageDisplay

For each panning cycle about 10¹³ recombinant phagemids in PBS (4 mMKH₂PO₄, 16 mM Na₂HPO₄, 115 mM NaCl, pH 7.4) were blocked with 2% (w/v)BSA in PBS/0.1T (PBS containing 0.1% (v/v) Tween 20 [polyoxyethylenesorbitan monolaurate; Sigma]) for 1 h in a total volume of 400 μl. 50 μlof a Streptavidin-coated magnetic particle suspension (StreptavidinMagnetic Particles; Roche Diagnostics, Mannheim, Germany) andNeutrAvidin-coated magnetic particles suspension (Sera-Mag Speed BeadsNeutrAvidin microparticles; Thermo Scientific, Fremont, Calif., USA),respectively were separately washed twice with PBS/0.1T and blocked with2% (w/v) BSA in PBS/0.1T for 1 h. Streptavidin-coated andNeutrAvidin-coated particles were applied alternately during theselection process to prevent enrichment of bead-specific phagemids.

The blocked Streptavidin/NeutrAvidin magnetic particles were thenincubated with 100 nM biotinylated Lu-DOTA-Bn in PBS/0.1T supplementedwith 2% BSA for 30 min under rotation, followed by blocking of freebiotin-binding sites via the addition of 0.1 mM D-Desthiobiotin (IBA,Göttingen, Germany) for 30 min. After three washing steps with 500 μlPBS/0.1T the blocked, target-loaded particles were incubated with 400 μlof the blocked phagemids under rotation for initially 2 h in the firstselection cycle. In the subsequent cycles, the incubation time waslowered to 1 h. Then, 0.1 mM D-Desthiobiotin was added to the mixture ofphagemids and target for 20 min, before the magnetic particles werepulled down with a single tube magnetic stand for 2 min. The supernatantcontaining unbound phagemids was discarded. The target/phagemidcomplexes bound to magnetic particles were washed 10 times with 500 μlPBS/0.1T containing 0.1 mM D-Desthiobiotin, and then bound phagemidswere eluted under conditions of competition by adding 400

100 μM non-biotinylated Lu-DOTA-Bn-NH₂ and rotation for 90 min. Intotal, four selection cycles were performed.

For amplification of eluted phagemids an exponentially growing cultureof E. coli XL-1 Blue was infected for 30 min at 37° C. and 140 rpm.After centrifugation (5 min, 4000 rpm, 4° C.) the bacterial pellet wasresuspended in 0.9 ml 2×YT medium (16 g/l Bacto Tryptone, 10 g/l BactoYeast Extract, 5 g/l NaCl, pH 7.5), plated onto LB-Amp plates (10 g/lBacto Tryptone, 5 g/l Bacto Yeast Extract, 5 g/l NaCl, 15 g/l BactoAgar, 100 mg/l ampicillin, pH 7.5), and incubated for 14-16 h at 32° C.Cells were then scraped off the plates and employed for rescue andre-amplification of the recombinant phagemids.

Screening of the enriched phagemid pools was performed by a screeningELISA (as illustrated in the description of FIG. 12) after the fourthpanning cycle.

Example 10 Selection of a1m Muteins with Affinity to Colchicine by PhageDisplay

Phage display for the selection of a1m muteins specific for biotinylatedColchicine from Example 6 was performed as described in Example 6 forthe Lu-DOTA-Bn target.

Example 11 Identification of a1m Muteins Specific for Lu-DOTA-Bn ViaScreening ELISA

After four cycles of phagemid selection with Lu-DOTA-Bn as described inExample 9, the enriched pool of a1m muteins was sub-cloned on theexpression plasmid pa1m2 (SEQ ID NO: 21), which encodes a fusion of theOmpA signal peptide for the periplasmic production in E. coli and thea1m coding region with the C-terminal Strep-tag II (Schmidt and Skerra(2007) Nat. Protoc. 2, 1528-1535) and used for transformation of the E.coli supE strain TG1/F⁻ (a derivative of E. coli K12 TG1 (Kim et al.(2009) J. Am. Chem. Soc. 131, 3565-3576), and subjected to a screeningELISA.

For this purpose, single randomly picked colonies from the enriched pooland wild-type a1m-expressing cells as negative control were grown in96-well plates (Multiple Well Plate 96 round bottom with lid; Sarstedt,Nuembrecht, Germany) in 100 μl TB-Amp medium (12 g/l Bacto Tryptone, 24g/l Bacto Yeast Extract, 55 mM glycerol, 17 mM KH₂PO₄, 72 mM K₂HPO₄, 100mg/l ampicillin) at 37° C., 300 rpm and 70% air humidity overnight(Minitron shaker, Infors AG, Bottmingen, Swiss). Next day, 5 μl of theovernight culture was used to inoculate a deep-well plate containing 700μl TB-Amp medium. Initially, cells were grown at 37° C., 300 rpm and 70%air humidity for 2 h, followed by 1.5 h incubation at 22° C. and 300 rpmuntil exponential phase was reached. Periplasmic expression of a1mmuteins was induced with 100 μl 0.2 μg/ml anhydrotetracycline (aTc;Acros, Geel, Belgium) dissolved in water-free dimethylformamide (DMF;Sigma-Aldrich, Steinheim, Germany) for 13-17 h at 20° C. and 300 rpm.Periplasmic protein extraction was performed with 200 μl BBS buffer (800mM Na-borate, 640 mM NaCl, 8 mM EDTA, pH 8.0) including 1 mg/ml lysozyme(AppliChem, Darmstadt, Germany) by incubation for 1 h at 4° C. andagitation at 900 rpm (Microplate Shaker, VWR International GmbH,Darmstadt, Germany). After blocking with 200 μl 10% (w/v) BSA inPBS/0.5T for 1 h at 4° C. and 900 rpm the plates were centrifuged for 30min at 4° C. and 5000 g. The supernatant was used for ELISA.

For capturing of biotinylated target, a 96-well MaxiSorp polystyrenemicrotiter plate (C96, Nunc, Langenselbold, Germany) was coated with 5μg/ml Streptavidin in PBS over night at 4° C. and blocked with 3% (w/v)BSA in TBS/0.1T (20 mM Tris, 2.5 mM KCl, 137 mM NaCl, pH 7.4 containing0.1% Tween) at room temperature for 1 h. After 5 washing steps withTBS/0.1T, 0.5 μM biotinylated Lu-DOTA-Bn from Example 6 was applied for1 h, followed by the addition 20 μl D-Desthiobiotin in a concentrationof 5 μM to prevent binding of a1m muteins to streptavidin via theirStrep-tag II. After the wells were washed, the cell extract from abovewas incubated for 1.5 h at room temperature. Bound a1m muteins weredetected via the Strep-tag II using a 1:1000 dilution ofStrep-MAB-Classic (IBA, Göttingen, Germany) in TBS/0.1T for 1 h. Thisprimary, mouse-derived antibody was then detected using an anti-mouseIgG (Fc-specific)/AP conjugate (Sigma) in a dilution of 1:2000 inTBS/0.1T as secondary antibody for 1 h. Final washing steps usingTBS/0.1T and TBS were followed by signal development in the presence of100 μl 0.5 mg/ml p-nitrophenyl phosphate (AppliChem, Darmstadt, Germany)in 100 mM Tris/HCl, pH 8.8, 100 mM NaCl, 5 mM MgCl₂ for up to 1.5 h.Absorption at 405 nm was measured in a SpectraMax 250 reader (MolecularDevices, Sunnyvale, USA).

Example 12 Identification of a1m Muteins Specific for Colchicine ViaScreening ELISA

a1m mutein with affinity to Colchicine were identified via screeningELISA as described in Example 11.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention. Theinvention has been described broadly and generically herein. Allpatents, patent applications, text books and peer-reviewed publicationsdescribed herein are hereby incorporated by reference in their entirety.Furthermore, where a definition or use of a term in a reference, whichis incorporated by reference herein is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply. Each of the narrower species and subgeneric groupingsfalling within the generic disclosure also form part of the invention.This includes the generic description of the invention with a proviso ornegative limitation removing any subject matter from the genus,regardless of whether or not the excised material is specificallyrecited herein. In addition, where features or aspects of the inventionare described in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Furtherembodiments of the invention will become apparent from the followingclaims.

The invention claimed is:
 1. A mutein of mature human alpha-1microglobulin (a1m) (SEQ ID NO: 1) comprising four peptide loopscomprising sequence positions 29-48, 63-80, 89-100, and 115-129,respectively, of the linear polypeptide sequence of mature human a1m(SEQ ID NO: 1), wherein at least one amino acid of each of at least twoof said four peptide loops has been mutated, wherein said mutein has atleast 80% sequence identity to the linear polypeptide sequence of maturehuman a1m (SEQ ID NO: 1), wherein said mutein binds a target other thanretinoic acid or retinol, and wherein said mutein has no detectablebinding affinity for retinoic acid or retinol.
 2. The mutein of claim 1,wherein at least one amino acid of each of at least three of said fourpeptide loops has been mutated.
 3. The mutein of claim 1, wherein atleast one amino acid of each of said four peptide loops has beenmutated.
 4. The mutein of claim 1, wherein at least one amino acid ofeach of at least three of the peptide loops corresponding to sequencepositions 63-80, 89-100, and 115-129 of the linear polypeptide sequenceof mature human a1m (SEQ ID NO: 1) has been mutated.
 5. The mutein ofclaim 1, wherein said mutein comprises at least one mutated amino acidat sequence positions 30, 47, 64, 73, 75, 77, 79, 90, 99, 116, and 128of the linear polypeptide sequence of mature human a1m (SEQ ID NO: 1).6. The mutein of claim 1, wherein said mutein comprises at least onemutated amino acid in each of the four loop regions comprising sequencepositions 32-46, 66-72, 91-98, and 118-126 of the linear polypeptidesequence of mature human a1m (SEQ ID NO: 1), respectively.
 7. The muteinof claim 1, wherein said mutein has no detectable binding affinity forretinoic acid or retinol, wherein the binding affinity is defined by adissociation constant and is detected using ELISA or using the techniqueof surface plasmon resonance.
 8. The mutein of claim 1, wherein saidmutein binds a target other than retinoic acid or retinol, wherein thebinding affinity is defined by a dissociation constant and is detectedusing ELISA or using the technique of surface plasmon resonance.
 9. Themutein of claim 1, wherein detectable binding affinity for retinoic acidor retinol is defined by a dissociation constant of at most 100 μM. 10.The mutein of claim 1, wherein the target is Lutetium (¹⁷⁷Lu) DOTA-TATEor Colchicine.
 11. The mutein of claim 10, wherein the mutein has atleast 90% sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 12-19 or a fragment or variant thereof.12. The mutein of claim 1, wherein the target is a tumor-specificcellular surface molecule.
 13. The mutein of claim 1, wherein the muteinis coupled to a compound selected from the group consisting of aprotein, an antibody, an enzyme, a radioactive moiety, and a moleculewith a defined binding characteristic.
 14. The mutein of claim 1,wherein the mutein is conjugated to a label selected from the groupconsisting of: organic molecules, enzyme labels, radioactive labels,colored labels, fluorescent labels, chromogenic labels, luminescentlabels, haptens, digoxigenin, biotin, metal complexes, metals, andcolloidal gold.
 15. The mutein of claim 1, wherein the mutein is fusedat its N-terminus and/or its C-terminus to a fusion partner.
 16. Themutein of claim 15, wherein the fusion partner extends the serumhalf-life of the mutein.
 17. The mutein of claim 16, wherein the fusionpartner that extends the serum half-life is selected from the groupconsisting of a polyalkylene glycol molecule, a polyethylene glycolmolecule, hydroxyethyl starch, a Fc part of an immunoglobulin, a CH3domain of an immunoglobulin, a CH4 domain of an immunoglobulin, analbumin binding peptide, and an albumin binding protein.
 18. Apharmaceutical composition comprising a mutein as defined in claim 1.19. A diagnostic or analytical kit comprising a mutein as defined inclaim
 1. 20. A mutein of claim 1, wherein said mutein has at least 85%sequence identity to the linear polypeptide sequence of mature human a1m(SEQ ID NO: 1).